Methods and compositions for treating autoimmune and allergic disorders

ABSTRACT

The current disclosure provides methods and compositions for treating autoimmune conditions, including allergic conditions such as food allergy. Certain aspects of the disclosure relate to methods for treatment of food allergy comprising administering a composition comprising  Phascolarctobacterium faecium  and/or  Ruminococcus bromii . In some aspects, disclosed are compositions comprising isolated, lyophilized bacteria such as  Phascolarctobacterium faecium  and/or  Ruminococcus bromii.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 63/055,227, filed Jul. 22, 2020, U.S. Provisional Patent ApplicationNo. 63/108,736, filed Nov. 2, 2020, and U.S. Provisional PatentApplication No. 63/122,833, filed Dec. 8, 2020, all of which areincorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numberAI134923, awarded by the National Institutes of Health. The governmenthas certain rights in this invention.

BACKGROUND I. Field

Aspects of this invention relate to the fields of molecular biology,immunology, and medicine.

II. Background

Recent surveys estimate that 32 million children and adults in theUnited States suffer from food allergies (1, 2). This marked increase inallergic responses to food in industrialized societies worldwideparallels increases in other non-communicable diseases (NCDs) includingobesity, diabetes, asthma, autism, and inflammatory bowel disease. TheseNCDs share an association with dysbiosis of the commensal microbiome,particularly in the gut (3). Early childhood is a particular period ofvulnerability for the maturation of the microbiota and the developingimmune system, which are intimately intertwined (4, 5).

An association between gut microbial community changes and childhoodfood allergies has been reported in some epidemiological studies (6-8).A Canadian healthy infant longitudinal development (CHILD) study showedalterations in the gut microbial community in food-sensitized infants,in which Enterobacteriaceae were overrepresented in a less diversemicrobial community in infants at 3 months of age, whereasBacteroidaceae were underrepresented at 1 year (8). A Chinese infantcohort study showed that infants with food allergies had a higherabundance of Firmicutes and lower abundance of Bacteroidetes at 6months, but no significant difference in the total microbial diversitywas found (7).

Oral (OIT) and epicutaneous (EPIT) immunotherapy, allergen-specificdesensitization protocols performed by introducing small but graduallyincreasing doses of allergen, have been shown to safely and effectivelydesensitize food-allergic patients to their allergens (9-11). However,OIT requires a prolonged period of updosing (usually years) during whichgastrointestinal symptoms are common and might contribute to the highwithdrawal rate observed in clinical trials (12,13). Although OIT canachieve short-term desensitization, this desensitization is notsustained without daily maintenance dosing, and long-term tolerance isnot induced in the majority of the cases (10). Livemicrobiome-modulating biotherapeutics have shown promise in clinicaltrials for a variety of diseases (14). Pre-clinical data suggests thatmicrobiome-modulating therapeutics may have the potential for improvingboth the efficacy and safety of OIT. Mouse model work has shown thatpreventing an allergic response to food requires the induction of both afood allergen-specific immunoregulatory response and a commensalbacteria-induced intestinal barrier protective response which regulatesepithelial permeability to food allergens (15).

Recognized is a need for compositions and methods for effective andsustained treatment of autoimmune conditions, including food allergy.

SUMMARY OF THE INVENTION

The current disclosure fulfills the need in the art by providing methodsand compositions for treating allergies, including food allergies, andother autoimmune conditions. Accordingly, aspects of the presentdisclosure provide methods and compositions useful for preventing orreducing an immune response to an allergen, including a food allergen.

Embodiments of the disclosure include methods for preventing an immuneresponse, methods for reducing an immune response, methods for treatingan allergy, methods for treating an autoimmune disorder, methods forreducing inflammation, methods for treating a food allergy, methods fordiagnosing a food allergy, methods for determining a risk of developinga food allergy, live bacterial compositions, freeze-dried bacterialcompositions, lyophilized bacterial compositions, and bacterialformulations.

Methods of the present disclosure can include at least 1, 2, 3, 4, 5, ormore of the following steps: administering a bacterial composition to asubject, administering one or more metabolites to a subject,administering one or more nanoparticles to a subject, administering oneor more microparticles to a subject, administering a prebiotic to asubject, determining a subject to have a decreased operational taxonomicunit (OTU) abundance score, determining a subject to have an increasedOTU abundance score, determining a subject to have a decreased abundanceof metabolites from a metabolic pathway, determining a subject to havean increased abundance of metabolites from a metabolic pathway,providing a food allergy therapy to a subject, diagnosing a food allergyin a subject, identifying a risk of a food allergy in a subject,sensitizing a subject to immunotherapy, treating a subject withimmunotherapy, immunizing a subject for a food allergy, generating abacterial composition, isolating one or more bacteria from a subject,purifying one or more bacteria, lyophilizing a bacterial product,obtaining a biological sample from a subject, obtaining a fecal samplefrom a subject, comparing a fecal sample from a healthy subject with afecal sample from an subject having a food allergy, generating abacterial product, and generating a mixture of two or more bacteria. Itis contemplated that any one or more of these steps may be excluded fromcertain embodiments of the disclosure.

Compositions of the present disclosure can include at least 1, 2, 3, ormore of the following components: a live bacterial product, afreeze-dried bacterial product, a lyophilized bacterial product, aprebiotic, Phascolarctobacterium faecium, Agathobaculum desmolans,Ruminococcus bromii, one or more nanoparticles, one or moremicroparticles, and a pharmaceutical excipient. It is contemplated thatany one or more of these components may be excluded from certainembodiments of the disclosure.

Aspects of the disclosure are directed to a method for preventing orreducing an immune response to an allergen in a subject, the methodcomprising administering to the subject a composition comprising atherapeutically effective amount of Phascolarctobacterium faecium and/orRuminococcus bromii. In some embodiments, the composition comprisesPhascolarctobacterium faecium. In some embodiments, the compositioncomprises between 1×10³ and 1×10¹⁵ colony forming units (CFU) ofPhascolarctobacterium faecium. In some embodiments, thePhascolarctobacterium faecium makes up at least 75%, 80%, 85%, 90%, 95%,or 99% of the composition, or any value or range derivable therein. Insome embodiments, the composition comprises Ruminococcus bromii. In someembodiments, the composition comprises between 1×10³ and 1×10¹⁵ CFU ofRuminococcus bromii. In some embodiments, the Ruminococcus bromii makesup at least 75%, 80%, 85%, 90%, 95%, or 99% of the composition, or anyvalue or range derivable therein. In some embodiments, the compositioncomprises Phascolarctobacterium faecium and Ruminococcus bromii. In someembodiments, the composition comprises at least 75%, 80%, 85%, 90%, 95%,or 99% Phascolarctobacterium faecium and Ruminococcus bromii, or more,or any value or range derivable therein. In some embodiments, thecomposition does not comprise more than a contaminating amount of anyother bacteria. In some embodiments, the composition does not comprise adetectable amount of any other bacteria. In some embodiments, thecomposition further comprises one or more microparticles. The bacteriamay be encapsulated in the one or more microparticles. Alternatively,the bacteria may not be encapsulated in the one or more microparticles.The bacteria may be associated with one or more nanoparticles.

In some embodiments, the allergen is a food allergen. In someembodiments, the food allergen is peanuts, tree nuts, shellfish, soy,egg, fish, mustard, oats, olives, corn, rice, pineapple, wheat, gluten,milk, sesame, garbanzo beans, bananas, kiwi, avocado, mangos, melons,carrots, cucumber, apples, squash, or crab. In some embodiments, thesubject was determined to have symptoms of a food allergy. In someembodiments, the food allergy is an allergy to 1, 2, 3, 4, 5, 6, 7, or 8different foods, or more. In some embodiments, the subject was diagnosedwith a food allergy. In some embodiments, the subject was not diagnosedwith a food allergy. In some embodiments, the subject has previouslybeen treated for a food allergy. In some embodiments, the subject wasdetermined to be resistant to the previous treatment. In someembodiments, the subject has not been previously treated for a foodallergy.

In some embodiments, the composition comprises a bacterial productcomprising the Phascolarctobacterium faecium and/or Ruminococcus bromii.In some embodiments, the bacterial product is a live bacterial product.In some embodiments, the bacterial product is a lyophilized orfreeze-dried bacterial product. In some embodiments, the bacterialproduct is isolated from a human subject. In some embodiments, thebacterial product is a bacterial product isolated from a non-humansubject. In some embodiments, the composition is administered orally.

In some embodiments, the method comprises preventing an anaphylacticresponse in the subject. In some embodiments, the method furthercomprises providing to the subject a prebiotic. The term “prebiotic”refers to an oligosaccharide or polysaccharide with a degree ofpolymerization of two or more that is not susceptible to digestion ordegradation prior to entering the upper gastrointestinal tract, such asthe small intestine, and is fermentable or digestible by microbes orother processes within the colon in which the fermented or digestedoligosaccharides or their byproducts of digestion alter the microbiomeor provide benefit to human or animal.

In some embodiments, the prebiotic comprises fructan (includingshort-chain fructooligosaccharides (scFOS), fructo-oligosaccharides(FOS), and inulin), galactans, glucans, and/or other oligosaccharides.Examples of such include short-chain FOS (shorter chains of fructosebelow, with degree of polymerization from 2-4); fructooligosaccharides(with degree of polymerization of 4-20); inulin (with degree ofpolymerization >20) or phlein; soybeanoligosaccharide (SOS);galactooligosacchararide (GOS); isomaltooligosaccharide (IMOS) (derivedfrom starches in wheat, barley, corn, oats, tapioca, rice, potato)including isomaltose, isomaltotriose, and panose;soybeanoligosaccharides (SOS) (from soybean) including raffinose andtetrasaccharide stachyose; xylooligosaccharides (XOS) including xylan,xylobiose, xylothiose, and xylotetraose (derived from starches found inbamboo shoots, fruits, vegetables, milk, and honey);pecticoligosaccharides (POS) including pectin; chitooligosaccharidesincluding chitin; lactulose; beta-glucans (from cereal grains, such asoat, barley, wheat, and rye); Type I resistant starch; Type II resistantstarch; and Type III resistant starch, which includes resistant starchthat is formed when starch-containing foods heated then are cooled (ex.pasta, potatoes, and rice). Further examples include polyols such asisomalt, maltitol, mannitol, sorbitol, xylitol, lactitol, erythritol,and polyglycitol. Sources of polyols include apples, apricots, avocados,blackberries, cherries, lychees, nectarines, peaches, pears, plums,prunes, watermelon, cauliflower, and mushrooms. Even further examplesinclude non-fructans such as dextrins, including maltodextrins,cyclodextrins, and pirodextrins (derived from potato and maize starch),wheat dextrin, high-amylose cornstarch (and maizestarch), amylose, andamylopectin. In some embodiments, the prebiotic comprises one or more ofwherein the prebiotic comprises one or more of galactooligosacchararide,lactulose, lactitol, erythritol, isomalt, polyglycitol, and succinate.In some embodiments, the prebiotic comprises one or more ofgalactooligosacchararide, lactulose, succinate, and lactitol.

In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13,14, or 15 grams of prebiotic, or more, is administered to the subject.In some embodiments, at least 10 grams of prebiotic is administered tothe subject. In some embodiments, the ratio or the colony forming unitsof the Phascolarctobacterium faecium or Ruminococcus bromii to grams orprebiotic is between 1000:1 and 10000:1, or any range or value derivabletherein.

Also included are derivatives and processed forms of the compoundsdescribed herein. Derivatives or processed forms may be modified toalter the fermentation properties of the prebiotic, for example, bymaking the prebiotic more readily digestible, more specific to a certaintype of bacterium, or to increase yield of fermentation products, suchas short-chain fatty acids (SCFAs) and/or other metabolites. Alsoincluded are foods and food derivatives known to contain quantities ofthese compounds and/or which are capable of having a prebiotic effect.These foods may be processed to isolate their starches, or may beadministered without isolation of their starches, e.g. by grinding thefood whole for consumption. Examples of such foods include: onion,artichoke, garlic, wheat, banana, asparagus, chicory, leek, tomato,bamboo shoots, fruits, vegetables, milk, honey, wheat, rye, barley,corn, oats, tapioca, rice, and potato.

In some embodiments, the method further comprises administering theallergen to the subject. In some embodiments, the allergen is a foodallergen. In some embodiments, the composition is administered prior tothe allergen. The composition may be administered at least or at most 4,6, 8, 10, 12, 16, 24, 36, 48, or 72 hours prior to the allergen, or anyrange or value derivable therein. In some embodiments, the compositionis administered after the allergen. The composition may be administeredat least or at most 4, 6, 8, 10, 12, 16, 24, 36, 48, or 72 hours afterto the allergen, or any range or value derivable therein.

In some embodiments, the method further comprises providing animmunotherapy to the subject. In some embodiments, the immunotherapy isan oral immunotherapy. In some embodiments, the immunotherapy is anepicutaneous immunotherapy. In some embodiments, the immunotherapy isprovided prior to administering the composition to the subject. In someembodiments, the immunotherapy is provided after administering thecomposition to the subject.

Further aspects of the disclosure relate to a method for treating foodallergy in a subject comprising providing a food allergy therapy (e.g.,an immunotherapy, a steroid, an antihistamine, a hormone, and/or amicrobiome-modulating therapy) where the subject was determined to havea food allergy. In some embodiments, the food allergy therapy is amicrobiome-modulating therapy. In some embodiments, themicrobiome-modulating therapy comprises a composition comprisingPhascolarctobacterium faecium and/or Ruminococcus bromii. In someembodiments, the subject was determined to have a decreased OTUabundance score relative to a control or reference sample, and whereinthe OTU abundance score was calculated using at least 20 of the OTUs ofTable 1. In some embodiments, the OTU abundance score was calculatedusing at least or at most, 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62,63, or 64 of the OTUs of Table 1. In some embodiments, the OTU abundancescore was calculated using one or more OTUs selected from 173135,174588, 174818, 176077, 176664, 178799, 186478, 188079, 189816, 190169,190649, 195258, 196139, 198184, 198941, 2331530, 269611, 295804, 343313,361702, 362342, 362765, 370099, 4402610, 509709, 556835, 574038, 658370,823634, New.CleanUp.ReferenceOTU110487, New.CleanUp.ReferenceOTU112566,New.CleanUp.ReferenceOTU122371, New.CleanUp.ReferenceOTU124061,New.CleanUp.ReferenceOTU127991, New.CleanUp.ReferenceOTU1320,New.CleanUp.ReferenceOTU134087, New.CleanUp.ReferenceOTU135990,New.CleanUp.ReferenceOTUl41755, New.CleanUp.ReferenceOTU141972,New.CleanUp.ReferenceOTUl49108, New.CleanUp.ReferenceOTU152821,New.CleanUp.ReferenceOTU153408, New.CleanUp.ReferenceOTU153961,New.CleanUp.ReferenceOTU156924, New.CleanUp.ReferenceOTU164203,New.CleanUp.ReferenceOTU28435, New.CleanUp.ReferenceOTU37938,New.CleanUp.ReferenceOTU41338, New.CleanUp.ReferenceOTU41725,New.CleanUp.ReferenceOTU47134, New.CleanUp.ReferenceOTU5050,New.CleanUp.ReferenceOTU57003, New.CleanUp.ReferenceOTU58395,New.CleanUp.ReferenceOTU58632, New.CleanUp.ReferenceOTU74051,New.CleanUp.ReferenceOTU79183, New.CleanUp.ReferenceOTU80284,New.CleanUp.ReferenceOTU86790, New.CleanUp.ReferenceOTU86928,New.CleanUp.ReferenceOTU89172, New.CleanUp.ReferenceOTU92834,New.CleanUp.ReferenceOTU97550, New.CleanUp.ReferenceOTU99007,New.ReferenceOTU129, and a combination thereof.

In some embodiments, the subject was determined to have a decreasedabundance of metabolites from a metabolic pathway, wherein the metabolicpathway is the diacylglycerol pathway, the sterol pathway, thetocopherol metabolism pathway, the gamma-glutamyl amino acid pathway, orthe endocannabinoid pathway. In some embodiments, the metabolic pathwayis the diacylglycerol pathway. In some embodiments, the subject wasdetermined to have a decreased abundance of linoleoyl-linoleoyl-glycerol(18:2/18:3) relative to a control or reference sample. In someembodiments, the subject was determined to have an increased abundanceof metabolites from a metabolic pathway, wherein the metabolic pathwayis the creatine metabolism pathway, the dihydroxy fatty acid pathway,the tyrosine metabolism pathway, or the food component/plant metabolismpathway. In some embodiments, the subject was determined to have anincreased abundance of secoisolariciresinol relative to a control toreference sample.

Further aspects relate to a method for diagnosing a subject with a foodallergy, the method comprising determining the subject to have adecreased OTU abundance score relative to a control or reference sample,wherein the OTU abundance score is calculated using at least 20 of theOTUs of Table 1. In some embodiments, the OTU abundance score iscalculated using at least or at most 20, 25, 30, 35, 40, 45, 50, 55, 60,61, 62, 63, or 64 of the OTUs of Table 1. In some embodiments, the OTUabundance score is calculated using one or more OTUs selected from173135, 174588, 174818, 176077, 176664, 178799, 186478, 188079, 189816,190169, 190649, 195258, 196139, 198184, 198941, 2331530, 269611, 295804,343313, 361702, 362342, 362765, 370099, 4402610, 509709, 556835, 574038,658370, 823634, New.CleanUp.ReferenceOTU110487,New.CleanUp.ReferenceOTU112566, New.CleanUp.ReferenceOTU122371,New.CleanUp.ReferenceOTU124061, New.CleanUp.ReferenceOTU127991,New.CleanUp.ReferenceOTU1320, New.CleanUp.ReferenceOTU134087,New.CleanUp.ReferenceOTU135990, New.CleanUp.ReferenceOTU141755,New.CleanUp.ReferenceOTUl41972, New.CleanUp.ReferenceOTU149108,New.CleanUp.ReferenceOTU152821, New.CleanUp.ReferenceOTU153408,New.CleanUp.ReferenceOTU153961, New.CleanUp.ReferenceOTU156924,New.CleanUp.ReferenceOTU164203, New.CleanUp.ReferenceOTU28435,New.CleanUp.ReferenceOTU37938, New.CleanUp.ReferenceOTU41338,New.CleanUp.ReferenceOTU41725, New.CleanUp.ReferenceOTU47134,New.CleanUp.ReferenceOTU5050, New.CleanUp.ReferenceOTU57003,New.CleanUp.ReferenceOTU58395, New.CleanUp.ReferenceOTU58632,New.CleanUp.ReferenceOTU74051, New.CleanUp.ReferenceOTU79183,New.CleanUp.ReferenceOTU80284, New.CleanUp.ReferenceOTU86790,New.CleanUp.ReferenceOTU86928, New.CleanUp.ReferenceOTU89172,New.CleanUp.ReferenceOTU92834, New.CleanUp.ReferenceOTU97550,New.CleanUp.ReferenceOTU99007, New.ReferenceOTU129, and a combinationthereof.

Yet further aspects of the disclosure are directed to methods fordiagnosing a subject with a food allergy, the method comprisingdetermining the subject to have a decreased or increased abundance ofmetabolites from a metabolic pathway associated with food allergy. Insome embodiments, the method comprises determining the subject to have adecreased abundance of metabolites from the diacylglycerol pathway, thesterol pathway, the tocopherol metabolism pathway, the gamma-glutamylamino acid pathway, and/or the endocannabinoid pathway. In someembodiments, the metabolic pathway is the diacylglycerol pathway. Insome embodiments, the subject was determined to have a decreasedabundance of linoleoyl-linoleoyl-glycerol (18:2/18:3). In someembodiments, the method comprises determining the subject to have anincreased abundance of metabolites from the creatine metabolism pathway,the dihydroxy fatty acid pathway, the tyrosine metabolism pathway,and/or the food component/plant metabolism pathway. In some embodiments,the subject was determined to have an increased abundance ofsecoisolariciresinol.

Additional aspects relate to a freeze-dried or lyophilized compositioncomprising Phascolarctobacterium faecium and Ruminococcus bromii. Insome embodiments, the composition does not comprise more than acontaminating amount of any other bacteria. In some embodiments, thecomposition does not comprise a detectable amount of any other bacteria.In some embodiments, the composition further comprises a pharmaceuticalexcipient. In some embodiments, the composition comprises one or moreadditional bacteria. In some embodiments, the composition is formulatedfor oral administration. Also provided are tablets, capsules, andpowders comprising a composition of the disclosure.

Further aspects are directed to a method for determining an OTUabundance score in a subject, the method comprising (a) obtaining afecal sample from the subject; (b) sequencing nucleic acid from thefecal sample; and (c) identifying OTUs in the fecal sample; wherein theOTUs comprise at least 20 of the OTUs of Table 1. In some embodiments,the OTUs comprise at least 20, at least 25, at least 30, at least 35, atleast 40, at least 45, at least 50, at least 55, at least 60, or more ofthe OTUs of Table 1. In some embodiments, the method further comprisescomparing the OTUs in the fecal sample with OTUs from a control orhealthy sample. In some embodiments, the method further comprisesidentifying the subject as having a food allergy if the OTU abundancescore is decreased relative to an OTU abundance score of a control orhealthy sample.

Yet further aspects are directed to a method for treating a subjectdetermined to have a decreased OTU abundance score relative to a controlor healthy sample, the method comprising providing to the subject acomposition comprising a therapeutically effective amount of (a)Phascolarctobacterium faecium or (b) Ruminococcus bromii, wherein theOTU abundance score was determined using at least 20 of the OTUs ofTable 1. In some embodiments, the OTUs comprise at least 20, at least25, at least 30, at least 35, at least 40, at least 45, at least 50, atleast 55, at least 60, or more of the OTUs of Table 1.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the measurement orquantitation method.

The use of the word “a” or “an” when used in conjunction with the term“comprising” may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

The phrase “and/or” means “and” or “or”. To illustrate, A, B, and/or Cincludes: A alone, B alone, C alone, a combination of A and B, acombination of A and C, a combination of B and C, or a combination of A,B, and C. In other words, “and/or” operates as an inclusive or.

The words “comprising” (and any form of comprising, such as “comprise”and “comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “includes” and“include”) or “containing” (and any form of containing, such as“contains” and “contain”) are inclusive or open-ended and do not excludeadditional, unrecited elements or method steps.

The compositions and methods for their use can “comprise,” “consistessentially of,” or “consist of” any of the ingredients or stepsdisclosed throughout the specification. Compositions and methods“consisting essentially of” any of the ingredients or steps disclosedlimits the scope of the claim to the specified materials or steps whichdo not materially affect the basic and novel characteristic of theclaimed invention. The phrase “consisting of” excludes any element,step, or ingredient not specified. It is contemplated that embodimentsdescribed in the context of the term “comprising” may also beimplemented in the context of the term “consisting of” or “consistingessentially of.”

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” when used in reference to a disease, disorder or medicalcondition, refer to therapeutic treatments for a condition, wherein theobject is to reverse, alleviate, ameliorate, inhibit, slow down or stopthe progression or severity of a symptom or condition. The term“treating” includes reducing or alleviating at least one adverse effector symptom of a condition. Treatment is generally “effective” if one ormore symptoms or clinical markers are reduced. For example, a treatmentfor food allergy is effective if one or more symptoms or clinicalmarkers of food allergy are reduced, such as a reduction in allergicsymptoms or immune markers. Alternatively, treatment is “effective” ifthe progression of a condition is reduced or halted. That is,“treatment” includes not just the improvement of symptoms or markers,but also a cessation or at least slowing of progress or worsening ofsymptoms that would be expected in the absence of treatment. Beneficialor desired clinical results include, but are not limited to, alleviationof one or more symptom(s), diminishment of extent of the deficit, and anincreased lifespan as compared to that expected in the absence oftreatment.

“Individual, “subject,” and “patient” are used interchangeably and canrefer to a human or non-human.

The term “isolated” encompasses a bacterium or other entity or substancethat has been (1) separated from at least some of the components withwhich it was associated when initially produced (whether in nature or inan experimental setting), and/or (2) produced, prepared, purified,and/or manufactured by the hand of man. Isolated bacteria may beseparated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or more of theother components with which they were initially associated. In someembodiments, isolated bacteria are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure. As usedherein, a substance is “pure” if it is substantially free of othercomponents.

The terms “purify,” “purifying” and “purified” refer to a bacterium orother material that has been separated from at least some of thecomponents with which it was associated either when initially producedor generated (e.g., whether in nature or in an experimental setting), orduring any time after its initial production. A bacterium or a bacterialpopulation may be considered purified if it is isolated at or afterproduction, such as from a material or environment containing thebacterium or bacterial population, and a purified bacterium or bacterialpopulation may contain other materials up to about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,or above about 90% and still be considered “isolated.” In someembodiments, purified bacteria and bacterial populations are more thanabout 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% pure. In the instance of bacterial compositions providedherein, the one or more bacterial types present in the composition canbe independently purified from one or more other bacteria producedand/or present in the material or environment containing the bacterialtype.

Any method in the context of a therapeutic, diagnostic, or physiologicpurpose or effect may also be described in “use” claim language such as“Use of” any compound, composition, or agent discussed herein forachieving or implementing a described therapeutic, diagnostic, orphysiologic purpose or effect.

Use of the one or more compositions may be employed based on any of themethods described herein. Other embodiments are discussed throughoutthis application. Any embodiment discussed with respect to one aspect ofthe disclosure applies to other aspects of the disclosure as well andvice versa. For example, any step in a method described herein can applyto any other method. Moreover, any method described herein may have anexclusion of any step or combination of steps. The embodiments in theExample section are understood to be embodiments that are applicable toall aspects of the technology described herein.

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventioncan be used to achieve methods of the invention.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows a schematic overview of the analysis workflow on themicrobial 16S sequencing data, metabolite profiling data, andintegration of the two types of data.

FIG. 2 shows a flow diagram of study design and participating patients.

FIGS. 3A-3E show an overview of microbial composition from healthy andallergic twins. FIG. 3A shows relative abundance of taxonomy at thefamily level. Sample IDs are shown on the x-axis (n=34); discordanttwins (12 pairs, n=24), for which one member was healthy and the othermember was allergic; concordant twins (5 pairs, n=10), for which bothmembers were allergic. Out of 36 total samples in the twin cohort, onesample (S5077) failed sequencing and yielded 0 reads, hence thecorresponding twin pair (#13) was excluded from 16S analysis. FIGS. 3Band 3C show correlation of OTU abundance between members from each twinpair, with the comparison between concordant and discordant twin pairsshown in FIG. 3B, and the comparison between dizygotic and monozygotictwins shown in FIG. 3C. Each dot denotes one twin pair (17 pairs shown).FIGS. 3D and 3E show Shannon alpha-diversity index between healthy andallergic groups; all samples are shown in FIG. 3D (n=34) and onlydiscordant twins shown in FIG. 3E (n=24). Each dot denotes one sample.In FIGS. 3B-3E, the bounds of the boxes represent the 25th and 75thpercentiles, the horizontal center line indicates the median, and thewhiskers extend to data points within a maximum of 1.5 times theinterquartile range (IQR). Two-sided Wilcoxon rank-sum test was used inFIGS. 3C-3D and two-sided Wilcoxon signed-rank test was used in FIG. 3E.

FIG. 4 shows a β-diversity Principal Coordinates Analysis (PCoA) of twinfecal microbial communities with weighted UniFrac measure. Shown is aplot of the first two principal coordinate axes (PC1 and PC2) explaining44.4% and 11.6% of the total variance among 34 samples from the healthyand allergic twins. Each dot represents one sample. Line connectssamples from the same twin pair. PERMANOVA was used to test thediversity differences between healthy and allergic groups (P=0.82).

FIG. 5 shows a binary heatmap of the 64 OTUs differentially abundantbetween healthy and allergic groups. Dark gray indicates the presence ofan OTU in a sample, and light grey indicates absence. The 64 OTUs arethose listed in Table 2. Out of 64 OTUs, 62 were more abundant in thehealthy group (healthy-abundant OTUs), and 2 are more abundant in theallergic group (allergic-abundant OTUs). OTU IDs are shown on the row inthe format of “OTU_ID|Family”, and those annotated with familyLachnospiraceae, Ruminococcaceae, or Clostridiales_unclassified arehighlighted. Sample IDs are shown on the column, with annotation barsabove the heatmap indicating concordant/discordant twin members, sex,and zygosity.

FIG. 6 shows a relative abundance heatmap of the 64 OTUs identifiedherein as differentially abundant. Of these 64 OTUs, 62 were moreabundant in the healthy group (healthy-abundant OTUs), and 2 were moreabundant in the allergic group (allergic-abundant OTU). OTU IDs areshown on the row in the format of “OTU_ID|Family,” and those annotatedwith the Clostridia class (Lachnospiraceae, Ruminococcaceae,unclassified Clostridiales) are highlighted. Sample IDs are shown on thecolumn, with annotation bars above the heatmap indicatingconcordant/discordant twin members, sex, and zygosity. Out of 36 samplestotal in the twin cohort, one sample (S5077) failed sequencing andyielded 0 reads, hence the corresponding twin pair (#13) was excludedfrom 16S analysis. DS-FDR was used on all samples (P<0.05) and two-sidedWilcoxon signed-rank test was used on discordant twin pairs (P<0.10),respectively. Unadjusted P-value thresholds were used to filter for OTUsof interest.

FIG. 7A shows a bubble plot showing the per-twin-pair abundancedifferences of the 64 differentially abundant OTUs. The size of eachcircle corresponds to the relative abundance of an OTU. Samples werearranged as: discordant twins (12 pairs, n=24), where one member ishealthy and the other member is allergic; concordant twins (5 pairs,n=10), where both members are allergic. OTU IDs are shown on the row inthe format of “OTU_ID|Family,” and those annotated with the Clostridiaclass (Lachnospiraceae, Ruminococcaceae, unclassified Clostridiales) arehighlighted. FIG. 7B shows the OTU abundance scores for each individual.The aggregated OTU abundance score was significantly higher in healthy(n=12) relative to allergic twins (n=22). The score was calculated usingthe 64 differentially abundant OTUs shown in FIG. 5 , FIG. 6 , FIG. 7Aand Table 2. Each dot denotes one sample. The bounds of the boxesrepresent the 25th and 75th percentiles, the horizontal center lineindicates the median, and the whiskers extend to data points within amaximum of 1.5 times the IQR. In FIG. 7A, DS-FDR was used on all samples(P<0.05) and two-sided Wilcoxon signed-rank test was used on discordanttwin pairs (P<0.10), respectively. Unadjusted P-value thresholds wereused to filter for OTUs of interest. In FIG. 7B, two-sided Wilcoxonrank-sum test was used on all samples.

FIG. 8 shows data demonstrating that the aggregated OTU abundance scoreis significantly higher in healthy relative to allergic group in thediscordant twin pairs (12 pairs, n=24). Each dot denotes one sample. Thebounds of the boxes represent the 25th and 75th percentiles, thehorizontal center line indicates the median, and the whiskers extend todata points within a maximum of 1.5 times the interquartile range (IQR).Two-sided Wilcoxon signed-rank test was used to compute the P-value.

FIG. 9 shows that test statistics of the differentially abundant OTUsfrom all samples (n=34) is correlated with that computed frommonozygotic twins only (n=28) comparing healthy and allergic groups.

FIGS. 10A and 10B show that the aggregated OTU abundance score remainssignificant in healthy relative to allergic group in monozygotic twins(n=28). FIG. 10A shows results from 28 samples from 14 pairs ofmonozygotic twins. FIG. 10B shows results from 18 samples from 9 pairsof monozygotic twins that are discordant for food allergy. Each dotdenotes one sample. The bounds of the boxes represent the 25th and 75thpercentiles, the horizontal center line indicates the median, and thewhiskers extend to data points within a maximum of 1.5 times the IQR.DS-FDR was used in FIG. 10A, two-sided Wilcoxon rank-sum test was usedin FIG. 10B.

FIG. 11 shows a Principle Component Analysis (PCoA) of fecalmetabolites. Shown is a plot of the first two principal component axes(PC1, PC2) explaining 21.66% and 8.54% of the total variance among 36samples from the healthy and allergic groups. One sample (S5077) and thecorresponding twin pair (#13) excluded from 16S analysis due to lowsequencing depth was included for metabolite analysis. Each dotrepresents one sample. Line connects samples from the same twin pair.PCA was performed on normalized and log 10-transformed quantification of1308 metabolites in total.

FIG. 12 shows that test statistics of the differentially abundantmetabolites from all samples (n=36) is correlated with that computedfrom monozygotic twins only (n=28) comparing healthy and allergicgroups. For each of the 97 metabolites differentially abundant betweenhealthy and allergic groups using all samples, test statistics wasre-computed using samples from monozygotic twins only. Two-sidedWilcoxon signed-rank test was used.

FIGS. 13A and 13B show data demonstrating that healthy and allergictwins exhibit differential enrichment in fecal metabolic pathways. FIG.13A shows that, of 36 samples, 33 metabolites were more abundant inhealthy (n=13) relative to allergic (n=23) group. Metabolites are shownon the row in the format of“COMP_ID|Biochemical_Name|Super_Pathway|Sub_Pathway”. Sample IDs areshown on the column, with annotation bars above the heatmap indicatingconcordant/discordant twin members, sex, and zygosity. FIG. 13B showsthat, of 36 samples, 64 metabolites were more abundant in allergic(n=23) relative to healthy (n=13) group. The same annotations are usedas in FIG. 13A. In FIGS. 13A and 13B, two-sided Welch's two-samplet-test was used on all samples (P<0.10) and unadjusted P-valuethresholds were used to filter for individual metabolites of interest.

FIGS. 14A-14C show that distinct metabolic pathways are enriched inhealthy and allergic twins. FIG. 14A shows that metabolites moreabundant in the healthy group or in the allergic group were enriched indifferent sub-pathways. Relative enrichment fold-change is shown on thex-axis, the name of sub-pathway is shown on the y-axis. P-value andFDR-adjusted P-value of each sub-pathway enrichment are shown next toeach horizontal bar. FIGS. 14B and 14C show representative examples ofmetabolites in the enriched sub pathways in the healthy or allergicgroup. FIG. 14B shows that the linoleoyl-linolenoyl-glycerol (18:2/18:3)[1]* (sub-pathway: Diacylglycerol) was higher in healthy (n=13) comparedto allergic (n=23) twin members. FIG. 14C shows that thesecoisolariciresinol (sub-pathway: Food Component/Plant) was higher inallergic twin pairs (n=23) compared to healthy twin pairs (n=13). Eachdot denotes one sample. The bounds of the boxes represent the 25th and75th percentiles, the horizontal center line indicates the median, andthe whiskers extend to data points within a maximum of 1.5 times theIQR. In FIG. 14A, hypergeometric test was used to compute the P-valuesof relative enrichment of metabolite sub-pathways and filtered byFDR-adjusted P<0.10. Pathways consisting of at least 2 significantmetabolites were included in the statistical test. After BH-FDR multipletesting correction, diacylglycerol (DAG) remained as the mostsignificantly enriched sub-pathway in metabolites more abundant inhealthy twins (FDR-adjusted P<0.00001). In FIGS. 14B and 14C, two-sidedWelch's two-sample t-test was used on all samples.

FIGS. 15A and 15B show representative examples of metabolitessignificantly higher in healthy relative to allergic group (as shown inFIGS. 14B and 14C) in the discordant twin pairs only (FIG. 15A), or viceversa (FIG. 15B) (13 pairs, n=26). Units shown on the y-axis in FIGS.15A and 15B represent the normalized raw area counts of UPLC MS/MSpeaks, rescaled to set the median equal to 1.00 for each biochemical.Each dot denotes one sample. The bounds of the boxes represent the 25thand 75th percentiles, the horizontal center line indicates the median,and the whiskers extend to data points within a maximum of 1.5 times theIQR. Two-sided paired t-test was used to compute the P-values.

FIGS. 16A-16H show the differential abundance of 32 metabolites betweenhealthy and allergic twin groups total (n=36) or in discordant twinpairs only (n=26 discordant pairs). Shown is a subset of the 97metabolites differentially abundant between healthy and allergic groupsacross all 36 samples. The one sample (S5077) and the corresponding twinpair (#13) excluded from 16S analysis due to low sequencing depth wasincluded for metabolite analysis, forming 13 discordant twin pairs. Fourmetabolites are shown per page. For each metabolite, two panels areshown: comparison between the two groups across all samples (left,n=36), and within discordant twin pairs only (right, 13 pairs, n=26),hence eight panels per page. P-values are shown in each panel. Forcomparison across all samples, two-sided Welch Two-Sample t-test wasused; for comparison within discordant twin pairs only, two-sided pairedt-test was used. All measure was normalized and log 10-transformedbefore statistical tests. The bounds of the boxes represent the 25th and75th percentiles, the horizontal center line indicates the median, andthe whiskers extend to data points within a maximum of 1.5 times theIQR.

FIG. 17 shows correlation between the 64 OTUs from FIG. 6 and the 97metabolites from FIGS. 13A and 13B. Metabolites are shown on the row inthe format of “COMP_ID|Biochemical_Name|Super_Pathway|Sub_Pathway”, andOTU IDs are shown on the column in the format of “OTU_ID|Family”.

FIG. 18 shows the OTUs differentially abundant between healthy andallergic groups that are correlated with different sets of metabolitesand pathways. Of 64 OTUs, 4 OTUs showed a strong correlation with the 97metabolites. The filtering of OTUs is illustrated in the analyticalworkflow (FIG. 1 ). Metabolites are shown on the row in the format of“COMP_ID|Biochemical_Name|Super_Pathway|Sub_Pathway”, and OTU IDs areshown on the column in the format of “OTU_ID|Family”. 3 OTUs that matchto bacteria species at >99% identity are bolded. OTUs were divided into4 clusters based on same height on the dendrogram shown on the columnusing R function cut.tree. Similarly, metabolites were divided into 5groups based on same height on the dendrogram shown on the row.Annotation to metabolite groups 1 to 5 were added based on thedistribution of Spearman's correlation coefficient ρ among thehealthy-abundant OTU clusters 1 to 3 consisting of 21 OTUs. Cluster 4only contains 1 OTU from allergic-abundant bacteria, hence was not usedfor metabolite group annotation. Spearman's correlation was used.

FIG. 19 shows distribution of metabolite Spearman's correlationcoefficient between healthy-abundant OTU clusters 1 to 3 for eachmetabolite group from FIG. 18 . Spearman's correlation coefficient ρfrom FIG. 18 is shown on the y-axis. Healthy-abundant OTU clusters 1 to3 from FIG. 18 are shown on the x-axis. Pairwise comparison P-values arecomputed between C1/C2, C1/C3, and C2/C3 for each metabolite group.Tukey's honestly significant difference (HSD) test was used whichcontrols false discovery rate for multiple comparisons. Each dot denotesone metabolite. The bounds of the boxes represent the 25th and 75thpercentiles, the horizontal center line indicates the median, and thewhiskers extend to data points within a maximum of 1.5 times the IQR.

FIGS. 20A-20E show that two bacterial species correlated with metabolicpathways are differentially abundant between healthy and allergic twins.FIG. 20A shows distribution of pathways in group 1 and 2 metabolites.Upper panel: super pathways in each group. The fraction of metabolitesfrom each super pathway on the y-axis was calculated by the number ofmetabolites that belong to this pathway divided by the total number ofmetabolites in a group. Bottom panels: number of metabolites that belongto each sub-pathway; (left panel) group 1, (right panel) group 2. FIG.20B shows that OTU 556835 (family Acidaminococcaceae) is significantlymore abundant in healthy compared to allergic group by 16S sequencing.This OTU was annotated as Phascolarctobacterium faecium at the specieslevel. FIG. 20C shows quantitative PCR (qPCR) validation of theabundance differences between healthy and allergic groups using P.faecium-specific primers. FIG. 20D shows that OTU188079 (familyRuminococcaceae) is significantly more abundant in healthy compared toallergic group by 16S sequencing. This OTU was annotated as Ruminococcusbromii at the species level. FIG. 20E shows qPCR validation of theabundance differences between healthy and allergic groups using R.bromii-specific primers. Units shown on the y-axis in FIG. 20C and FIG.20E represent 2^(−Ct) normalized to total 16S rRNA copies per gram offecal material and multiplied by a constant (1×10²²) to bring all valuesabove 1. In FIGS. 20B-20E, n=30 samples (15 twin pairs) with DNAavailable for qPCR validation are shown (10 healthy, 20 allergic). Eachdot denotes one sample. The bounds of the boxes represent the 25th and75th percentiles, the horizontal center line indicates the median, andthe whiskers extend to data points within a maximum of 1.5 times theIQR. DS-FDR was used in FIG. 20B and FIG. 20D. In FIG. 20C and FIG. 20E,qPCR data were log 10 transformed, and two-sided Wilcoxon rank-sum testwas used.

FIGS. 21A and 21B show qPCR validation of Phascolarctobacterium faeciumdiscovered by 16S sequencing platform, shown in discordant twin pairsonly (10 pairs, n=20). 2 out of 12 discordant pairs did not have DNAmaterial left for qPCR validation and were not included. FIG. 21A showsthat OTU 556835 (family Acidaminococcaceae) is significantly moreabundant in healthy compared to allergic group by 16S sequencing. ThisOTU was annotated as Phascolarctobacterium faecium at the species levelwith 99% sequence identity (NCBI accession ID NR_026111.1). P-value wasre-calculated amongst the 10 twin pairs shown here from the 16Ssequencing data, instead of 12 twin pairs total. FIG. 21B showsquantitative PCR (qPCR) validation of the abundance differences betweenhealthy and allergic groups using P. faecium-specific primers. Unitsshown on the y-axis in represent 2^(−Ct) normalized to total 16S rRNAcopies per gram of fecal material and multiplied by a constant (1×10²²)to bring all values above 1. Samples with abundance above the detectionlimit in both platforms are shown. Each dot denotes one sample. Thebounds of the boxes represent the 25th and 75th percentiles, thehorizontal center line indicates the median, and the whiskers extend todata points within a maximum of 1.5 times the IQR. Two-sided Wilcoxonsigned-rank test was used in FIGS. 21A and 21B. qPCR data in FIG. 21Bwere log 10 transformed before statistical testing.

FIGS. 22A and 22B show qPCR validation of Ruminococcus bromii discoveredby 16S sequencing platform, shown in discordant twin pairs only (10pairs, n=20). 2 out of 12 discordant pairs did not have DNA materialleft for qPCR validation and were not included. FIG. 22A shows thatOTU188079 (family Ruminococcaceae) is significantly more abundant inhealthy compared to allergic group by 16S sequencing. This OTU wasannotated as Ruminococcus bromii at the species level with 99% sequenceidentity (NCBI accession ID NR_025930.1). P-value was re-calculatedamongst the 10 twin pairs shown here from the 16S sequencing data,instead of 12 twin pairs total. FIG. 22B shows qPCR validation of theabundance differences between healthy and allergic groups using R.bromii-specific primers. Units shown on the y-axis represent 2^(−Ct)normalized to total 16S rRNA copies per gram of fecal material andmultiplied by a constant (1×10²²) to bring all values above 1. Sampleswith abundance above the detection limit in both platforms are shown.Each dot denotes one metabolite. The bounds of the boxes represent the25th and 75th percentiles, the horizontal center line indicates themedian, and the whiskers extend to data points within a maximum of 1.5times the IQR. Two-sided Wilcoxon signed-rank test was used in FIGS. 22Aand 22B. qPCR data in FIG. 22B were log 10 transformed beforestatistical testing.

DETAILED DESCRIPTION

Toward the goal of developing novel microbiome-modulating therapeutics,the inventors identified bacterial taxa, and their products, associatedwith a healthy microbiota. Fecal samples were examined from a uniquecohort of food allergic and healthy twins across a broad age range and adistinct set of bacterial species and metabolites were identified thatdistinguished the healthy and allergic groups. A unique microbiotasignature was identified consisting of 64 operational taxonomic units(OTUs), 62 healthy-abundant and 2 allergic-abundant, that weresignificantly different between the two groups. By integratingmicrobiota and metabolite abundance, a significant enrichment wasidentified in healthy twins in particular metabolite pathways not seenin their allergic counterparts, particularly diacylglycerol, anessential microbially-derived, lipid second messenger involved innumerous cell signaling cascades supporting biosynthesis ofglycerolipids and regulating protein kinase C (16). The inventors alsoidentified two bacterial species more abundant in healthy twins whichcorrelate with differentially abundant metabolites:Phascolarctobacterium faecium, an acetate/propionate-producing obligateanaerobe associated with increased diacylglycerol and biotin metabolism(17, 18) and Ruminococcus bromii, a keystone resistant-starch(RS)-degrading strict anaerobe associated with fatty acid, sterol, andamino acid metabolism (19, 20). These findings demonstrate bacteria andmetabolites which may be useful for therapeutic indications in foodallergy.

I. Microbial Compositions

Embodiments of the present disclosure concern microbial compositions forthe treatment of allergic disease, including food allergy.

The present disclosure also provides pharmaceutical compositionscomprising one or more microbial cultures. The bacterial speciestherefore are present in the dose form as live bacteria, whether indried or lyophilized form. This may be adapted for suitableadministration; for example, in tablet or powder form, in some caseswith an enteric coating, for oral treatment.

In some embodiments, the composition is formulated for oraladministration. Oral administration may be achieved using a chewableformulation, a dissolving formulation, an encapsulated/coatedformulation, a multi-layered lozenge (to separate active ingredientsand/or active ingredients and excipients), a slow release/timed releaseformulation, or other suitable formulations known to persons skilled inthe art. Although the word “tablet” is used herein, the formulation maytake a variety of physical forms that may commonly be referred to byother terms, such as lozenge, pill, capsule, or the like.

While the compositions of the present disclosure may be formulated fororal administration, other routes of administration can be employed,however, including, but not limited to, subcutaneous, intramuscular,intradermal, transdermal, intraocular, intraperitoneal, mucosal,vaginal, rectal, and intravenous.

The desired dose of the composition of the present disclosure may bepresented in multiple (e.g., two, three, four, five, six, or more)sub-doses administered at appropriate intervals throughout the day,week, month or year.

In one aspect, the disclosed composition is prepared as a capsule. Thecapsule may be a hollow, generally cylindrical capsule formed fromvarious substances, such as gelatin, cellulose, carbohydrate or thelike.

In another aspect, the disclosed composition is prepared as asuppository. The suppository may include but is not limited to thebacteria and one or more carriers, such as polyethylene glycol, acacia,acetylated monoglycerides, carnuba wax, cellulose acetate phthalate,corn starch, dibutyl phthalate, docusate sodium, gelatin, glycerin, ironoxides, kaolin, lactose, magnesium stearate, methyl paraben,pharmaceutical glaze, povidone, propyl paraben, sodium benzoate,sorbitan monoleate, sucrose talc, titanium dioxide, white wax andcoloring agents.

In some aspects, the disclosed microbial composition is prepared as atablet. The tablet may include the bacteria and one or more tabletingagents, such as dibasic calcium phosphate, stearic acid, croscarmellose,silica, cellulose and cellulose coating. The tablets may be formed usinga direct compression process, though those skilled in the art willappreciate that various techniques may be used to form the tablets.

In other aspects, the disclosed microbial composition is formed as foodor drink or, alternatively, as an additive to food or drink, wherein anappropriate quantity of bacteria is added to the food or drink to renderthe food or drink the carrier.

In some embodiments, the microbial composition may further comprise afood or a nutritional supplement effective to stimulate the growth ofbacteria (e.g., Phascolarctobacterium faecium, Ruminococcus bromii,and/or Agathobaculum desmolans) present in the gastrointestinal tract ofthe subject. In some embodiments, the nutritional supplement is producedby another bacterium associated with a healthy human gut microbiome.

In some embodiments, the disclosed microbial compositions comprise oneor more microparticles. In some embodiments, the compositions compriseone or more bacteria (e.g., Phascolarctobacterium faecium, Ruminococcusbromii, and/or Agathobaculum desmolans) encapsulated withinmicroparticles. Microparticles may be any type of microparticlessuitable for providing a microbial composition to a subject. In someembodiments, microparticles are lipid microparticles.

In some embodiments, a microbial composition comprises an isolatedbacteria selected from Phascolarctobacterium faecium, Ruminococcusbromii and Agathobaculum desmolans. In some embodiments, a microbialcomposition comprises Phascolarctobacterium faecium and Ruminococcusbromii. In some embodiments, a microbial composition comprisesPhascolarctobacterium faecium and Agathobaculum desmolans. In someembodiments, a microbial composition comprises Agathobaculum desmolansand Ruminococcus bromii. In some embodiments, a microbial compositioncomprises a minimum amount of one or more isolated bacteria. Forexample, a microbial composition may comprise at least, 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of a particular type orparticular types of bacteria (e.g., Phascolarctobacterium faecium,Ruminococcus bromii, and/or Agathobaculum desmolans), or any value orrange derivable therein. In some embodiments, a microbial compositioncomprises at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9% of Phascolarctobacterium faecium, or any value or range derivabletherein. In some embodiments, a microbial composition comprises at least%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of Agathobaculumdesmolans, or any value or range derivable therein. In some embodiments,a microbial composition comprises at least %, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, or 99.9% of Ruminococcus bromii, or any value or rangederivable therein. In some embodiments, a microbial compositioncomprises at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or99.9% of Phascolarctobacterium faecium and Agathobaculum desmolans, orany value or range derivable therein. In some embodiments, a microbialcomposition comprises at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, or 99.9% of Phascolarctobacterium faecium and Ruminococcusbromii, or any value or range derivable therein. In some embodiments, amicrobial composition comprises at least 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 8100, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, or 99.9% of Ruminococcus bromii and Agathobaculumdesmolans, or any value or range derivable therein.

In some embodiments, a microbial composition comprises, in addition toPhascolarctobacterium faecium, Ruminococcus bromii, and/or Agathobaculumdesmolans, one or more additional bacteria. In some embodiments, amicrobial composition does not comprise a detectable amount of one ormore additional bacteria. In some embodiments, a microbial compositiondoes not comprise more than a contaminating amount of one or moreadditional bacteria. A contaminating amount of bacteria in a compositionmay be at most 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001%, or 0.0001% ofthe composition, or any range or value derivable therein.

II. Sample Preparation

In certain aspects, methods involve obtaining a sample from a subject(also a “biological sample”). The methods of obtaining provided hereinmay include methods of biopsy such as fine needle aspiration, coreneedle biopsy, vacuum assisted biopsy, incisional biopsy, excisionalbiopsy, punch biopsy, shave biopsy or skin biopsy. In other embodimentsthe sample may be obtained from any of the tissues provided herein thatinclude but are not limited to non-cancerous or cancerous tissue andnon-cancerous or cancerous tissue from the serum, gall bladder, mucosal,skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney,smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, orthyroid tissue. Alternatively, the sample may be obtained from any othersource including but not limited to blood, sweat, hair follicle, buccaltissue, tears, menses, feces, or saliva. In certain aspects of thecurrent methods, any medical professional such as a doctor, nurse ormedical technician may obtain a biological sample for testing. Yetfurther, the biological sample can be obtained without the assistance ofa medical professional.

A sample may include but is not limited to, tissue, cells, or biologicalmaterial from cells or derived from cells of a subject. The biologicalsample may be a heterogeneous or homogeneous population of cells ortissues. The biological sample may be obtained using any method known tothe art that can provide a sample suitable for the analytical methodsdescribed herein. The sample may be obtained by non-invasive methodsincluding but not limited to: scraping of the skin or cervix, swabbingof the cheek, saliva collection, urine collection, feces collection,collection of menses, tears, or semen.

The sample may be obtained by methods known in the art. In certainembodiments the samples are obtained by biopsy. In other embodiments thesample is obtained by swabbing, endoscopy, scraping, phlebotomy, or anyother methods known in the art. In some cases, the sample may beobtained, stored, or transported using components of a kit of thepresent methods. In some cases, multiple samples, such as multiple fecalsamples may be obtained for diagnosis by the methods described herein.In other cases, multiple samples, such as one or more samples of onetype (for example fecal) and one or more samples of another type (forexample blood) may be obtained for diagnosis by the methods. In somecases, multiple samples may be obtained at the same or different times.Samples may be obtained at different times are stored and/or analyzed bydifferent methods. For example, a sample may be obtained and analyzed byroutine staining methods or any other cytological analysis methods.

In some embodiments the biological sample may be obtained by aphysician, nurse, or other medical professional such as a medicaltechnician, endocrinologist, cytologist, phlebotomist, radiologist, or apulmonologist. The medical professional may indicate the appropriatetest or assay to perform on the sample. In certain aspects a molecularprofiling business may consult on which assays or tests are mostappropriately indicated. In further aspects of the current methods, thepatient or subject may obtain a biological sample for testing withoutthe assistance of a medical professional, such as obtaining a wholeblood sample, a urine sample, a fecal sample, a buccal sample, or asaliva sample.

In other cases, the sample is obtained by an invasive procedureincluding but not limited to: biopsy, needle aspiration, endoscopy, orphlebotomy. The method of needle aspiration may further include fineneedle aspiration, core needle biopsy, vacuum assisted biopsy, or largecore biopsy. In some embodiments, multiple samples may be obtained bythe methods herein to ensure a sufficient amount of biological material.

General methods for obtaining biological samples are also known in theart. Publications such as Ramzy, Ibrahim Clinical Cytopathology andAspiration Biopsy 2001, which is herein incorporated by reference in itsentirety, describes general methods for biopsy and cytological methods.In one embodiment, the sample is a fecal sample. In some cases, the fineneedle aspirate sampling procedure may be guided by the use of anultrasound, X-ray, or other imaging device.

In some embodiments of the present methods, the molecular profilingbusiness may obtain the biological sample from a subject directly, froma medical professional, from a third party, or from a kit provided by amolecular profiling business or a third party. In some cases, thebiological sample may be obtained by the molecular profiling businessafter the subject, a medical professional, or a third party acquires andsends the biological sample to the molecular profiling business. In somecases, the molecular profiling business may provide suitable containers,and excipients for storage and transport of the biological sample to themolecular profiling business.

In some embodiments of the methods described herein, a medicalprofessional need not be involved in the initial diagnosis or sampleacquisition. An individual may alternatively obtain a sample through theuse of an over the counter (OTC) kit. An OTC kit may contain a means forobtaining said sample as described herein, a means for storing saidsample for inspection, and instructions for proper use of the kit. Insome cases, molecular profiling services are included in the price forpurchase of the kit. In other cases, the molecular profiling servicesare billed separately. A sample suitable for use by the molecularprofiling business may be any material containing tissues, cells,nucleic acids, genes, gene fragments, expression products, geneexpression products, or gene expression product fragments of anindividual to be tested. Methods for determining sample suitabilityand/or adequacy are provided.

In some embodiments, the subject may be referred to a specialist such asan oncologist, surgeon, endocrinologist, or gastroenterologist. Thespecialist may likewise obtain a biological sample for testing or referthe individual to a testing center or laboratory for submission of thebiological sample. In some cases the medical professional may refer thesubject to a testing center or laboratory for submission of thebiological sample. In other cases, the subject may provide the sample.In some cases, a molecular profiling business may obtain the sample.

III. Administration of Therapeutic Compositions

Embodiments of the disclosure relate to compositions and methodscomprising therapeutic compositions. Different therapies may beadministered in one composition or in more than one composition, such as2 compositions, 3 compositions, or 4 compositions. Various combinationsof the agents may be employed.

In some embodiments, the therapy provided herein comprisesadministration of a combination of therapeutic agents, such as acombination of two or more microbial compositions. In some embodiments,the therapy comprises administration of a combination or one or moremicrobial compositions and a prebiotic. In some embodiments, the therapycomprises administration of a combination of one or more microbialcompositions and an allergen. For example, a therapy may compriseadministration of one or more bacteria and a food of a food allergy(e.g., administration of bacteria and a peanut product to an individualwith a peanut allergy). The therapy may be administered in any suitablemanner known in the art. For example, a microbial composition, aprebiotic, and/or an allergen may be administered sequentially (atdifferent times) or concurrently (at the same time).

Embodiments of the disclosure relate to compositions and methodscomprising bacteria, a food allergen, and/or one or more prebiotics. Thebacteria, allergen, and/or prebiotic(s) may be administered in onecomposition or in more than one composition, such as 2 compositions, 3compositions, or 4 compositions. Various combinations of the agents maybe employed, for example, a bacterium (or composition comprisingbacteria) is “A” and a prebiotic or an allergen is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

In some embodiments, the microbial composition is administered prior tothe prebiotic. In some embodiments, the microbial composition isadministered at least, at most, or about 1, 2, 3, 5, 6, 12, 24 hours or2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or anyderivable range therein) prior to the prebiotic. In some embodiments, atleast 1, 2, 3, 4, 5, 6, or 7 doses (or any derivable range therein) ofthe microbial composition is administered at least, at most, or about 1,2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7,or 8 weeks (or any derivable range therein) prior to the prebiotic. Insome embodiments, the microbial composition is administered after theprebiotic. In some embodiments, the microbial composition isadministered at least, at most, or about 1, 2, 3, 5, 6, 12, 24 hours or2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or anyderivable range therein) after the prebiotic or after at least one ofthe prebiotics or after at least 2 of the prebiotics. In someembodiments, at least 1, 2, 3, 4, 5, 6, or 7 doses (or any derivablerange therein) of the microbial composition is administered at least, atmost, or about 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable range therein) after theprebiotic or after at least one of the prebiotics or after at least 2 ofthe prebiotics.

In some embodiments, the microbial composition is administered prior tothe allergen. In some embodiments, the microbial composition isadministered at least, at most, or about 1, 2, 3, 5, 6, 12, 24 hours or2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or anyderivable range therein) prior to the allergen. In some embodiments, atleast 1, 2, 3, 4, 5, 6, or 7 doses (or any derivable range therein) ofthe microbial composition is administered at least, at most, or about 1,2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7,or 8 weeks (or any derivable range therein) prior to the allergen. Insome embodiments, the microbial composition is administered after theallergen. In some embodiments, the microbial composition is administeredat least, at most, or about 1, 2, 3, 5, 6, 12, 24 hours or 2, 3, 4, 6,8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks (or any derivable rangetherein) after the allergen. In some embodiments, at least 1, 2, 3, 4,5, 6, or 7 doses (or any derivable range therein) of the microbialcomposition is administered at least, at most, or about 1, 2, 3, 5, 6,12, 24 hours or 2, 3, 4, 6, 8, 10, days or 2, 3, 4, 5, 6, 7, or 8 weeks(or any derivable range therein) after the allergen.

In some embodiments, the microbial modulator composition is formulatedfor oral administration. The skilled artisan knows a variety of formulaswhich can encompass living or killed microorganisms and which canpresent as food supplements (e.g., pills, tablets, powders, and thelike) or as functional food such as drinks or fermented yogurts.

The agents of the disclosure may be administered by the same route ofadministration or by different routes of administration. In someembodiments, the prebiotic is administered intravenously,intramuscularly, subcutaneously, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. In some embodiments,the microbial composition is administered intravenously,intramuscularly, subcutaneously, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. The appropriatedosage may be determined based on the type of disease to be treated,severity and course of the disease, the clinical condition of theindividual, the individual's clinical history and response to thetreatment, and the discretion of the attending physician.

In some embodiments, a composition may comprise a therapeuticallyeffective amount of one or more bacteria. As used here, a“therapeutically effective” amount of a bacterium describes an amountsufficient to be effective in treating a desired condition, for examplefood allergy. In some embodiments, a therapeutically effective amount ofeach of the at least one isolated or purified population of bacteria oreach of the at least two, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, or 15isolated or purified populations of bacteria that is administered to ahuman will be at least about 1×10³ colony forming units (CFU) ofbacteria or at least about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹,1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵ CFU (or any derivablerange therein). In some embodiments, a single dose will contain bacteria(such as a specific bacteria or species, genus, or family describedherein) present in an amount of least, at most, or about 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³,1×10¹⁴, 1×10¹⁵ or more CFU (or any derivable range therein). In someembodiments, a single dose will contain at least, at most, or about1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹²,1×10¹³, 1×10¹⁴, 1×10¹⁵ or greater than 1×10¹⁵ CFU (or any derivablerange therein) of total bacteria.

In some embodiments, a therapeutically effective amount of each of theat least one isolated or purified population of bacteria that isadministered to a human will be at least about 1×10³ cells of bacteriaor at least about 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴, 1×10¹⁵ cells (or any derivable rangetherein). In some embodiments, a single dose will contain bacteria (suchas a specific bacteria or species, genus, or family described herein)present in an amount of at least, at most, or about 1×10³, 1×10⁴, 1×10⁵,1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10¹², 1×10¹³, 1×10¹⁴,1×10¹⁵ or more cells (or any derivable range therein). In someembodiments, a single dose will contain at least, at most, or about1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹, 1×10²,1×10¹³, 1×10¹⁴, 1×10¹⁵ or greater than 1×10¹⁵ cells (or any derivablerange therein) of total bacteria.

Embodiments of the disclosure relate to compositions and methodscomprising one or more metabolites. In some embodiments, disclosed arecompositions comprising one or more metabolites from a metabolicpathway. In some embodiments, the metabolic pathway is thediacylglycerol pathway, the biotin metabolism pathway, the sterolpathway, the tocopherol metabolism pathway, the gamma-glutamyl aminoacid pathway, or the endocannabinoid pathway. In some embodiments, thedisclosed compositions comprise one or more metabolites from thediacylglycerol pathway. In some embodiments, the disclosed compositionscomprise linoleoyl-linoleoyl-glycerol (18:2/18:3). In some embodiments,the disclosed compositions comprise diacylglycerol. In some embodiments,the metabolic pathway is the creatine metabolism pathway, the dihydroxyfatty acid pathway, the cardiovascular drug metabolism pathway, thetyrosine metabolism pathway, or the food component/plant metabolismpathway. In some embodiments, the disclosed compositions comprisesecoisolariciresinol. Also contemplated are compositions comprisinginhibitors or activators of one of more metabolic pathways disclosedabove. For example, in some embodiments, a composition comprises anactivator of the diacylglycerol pathway, the biotin metabolism pathway,the sterol pathway, the tocopherol metabolism pathway, thegamma-glutamyl amino acid pathway, or the endocannabinoid pathway. Insome embodiments, a composition comprises an activator of thediacylglycerol pathway. In some embodiments, a composition comprises aninhibitor of the creatine metabolism pathway, the hydroxy fatty acidpathway, the cardiovascular drug metabolism pathway, the tyrosinemetabolism pathway, or the food component/plant metabolism pathway. Insome embodiments, a composition comprises an inhibitor of the creatinemetabolism pathway.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined-quantity of the therapeutic composition. Thequantity to be administered, and the particular route and formulation,is within the skill of determination of those in the clinical arts. Aunit dose need not be administered as a single injection but maycomprise continuous infusion over a set period of time. In someembodiments, a unit dose comprises a single administrable dose.

Precise amounts of the therapeutic composition also depend on thejudgment of the practitioner and are peculiar to each individual.Factors affecting dose include physical and clinical state of thepatient, the route of administration, the intended goal of treatment(alleviation of symptoms versus cure) and the potency, stability andtoxicity of the particular therapeutic substance or other therapies asubject may be undergoing.

Prebiotics may be formulated using techniques of pharmaceuticalformulation known in the art. They may be formulated using specializedtechniques known for delivery to specific regions of thegastrointestinal tract. Two examples known in the art are described inpublished PCT application WO 2018/195067 A1 and also in US Patentapplication publication US 2017/0209504 A1, each of which areincorporated by reference. Other formulations for prebiotics orcombinations of prebiotics and bacteria (e.g., Phascolarctobacteriumfaecium, Ruminococcus bromii, and/or Agathobaculum desmolans) are knownin the art. In some embodiments, the compositions of the disclosureinclude a compound carrying butyrate or other microbial metabolites,such as those described in WO 2018/195067. IV. Methods of Treatment

The methods of the disclosure relate to the treatment of inflammatory,autoimmune, or allergic disease. In some embodiments, the methods andcompositions are for the treatment of a food allergy. In someembodiments, the food allergy is an allergy to one or more of peanuts,tree nuts, shellfish, soy, egg, fish, mustard, oats, olives, corn, rice,pineapple, wheat, gluten, milk, sesame, garbanzo beans, bananas, kiwi,avocado, mangos, melons, carrots, cucumber, apples, squash, and crab. Insome embodiments, the disclosed methods comprise treatment of a subjecthaving an allergy to a single food. In some embodiments, the disclosedmethods comprise treatment of a subject having allergies to multiplefoods (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 foods, or more). In someembodiments, subjects treated by the disclosed methods have beendiagnosed with a food allergy. In some embodiments, subjects treated bythe disclosed methods have not been diagnosed with a food allergy. Insome embodiments, subjects treated by the disclosed methods are at riskfor having or developing an allergy to one or more foods.

In some embodiments, the disclosed methods comprise treatment of asubject following identification and/or analysis of one or moreoperational taxonomic units (OTUs). OTUs describe clusters of similarsequence results (e.g., from sequencing of 16S rRNA from a biologicalsample comprising a plurality of bacteria) based on a sequencesimilarity threshold (e.g., 97% similar, 98% similar, or 99% similar).OTUs may be further characterized to determine the closest matchinggenus or species. OTUs may be used to identify a subject as having afood allergy and/or inform a food allergy treatment decision. Examplesof OTUs which may be used in the methods of the present disclosure areprovided in Table 1.

TABLE 1 List of 64 OTUs identified herein as differentially abundantbetween healthy and allergic twins. OTU Taxonomy 173135 k _(—) Bacteria;p _(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g_; s_(—) 174588 k _(—) Bacteria; p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)174818 k _(—) Bacteria; p _(—) Firmicutes; c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g_; s_(—) 176077 k _(—) Bacteria;p _(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f_; g_;s_(—) 176664 k _(—) Bacteria; p _(—) Firmicutes; c _(—) Clostridia; o_(—) Clostridiales; f _(—) Lachnospiraceae; g _(—) Roseburia; s _(—)faecis 178799 k _(—) Bacteria; p _(—) Firmicutes; c _(—) Clostridia; o_(—) Clostridiales; f _(—) Ruminococcaceae; g_; s_(—) 186478 k _(—)Bacteria; p _(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f_(—) Ruminococcaceae; g_; s_(—) 188079 k _(—) Bacteria; p _(—)Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g _(—) Ruminococcus 189816 k _(—) Bacteria; p _(—)Firmicutes; c _(—) Clostridia; o _(—) Clostridiales 190169 k _(—)Bacteria; p _(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f_(—) Ruminococcaceae; g_; s_(—) 190649 k _(—) Bacteria; p _(—)Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g_; s_(—) 195258 k _(—) Bacteria; p _(—) Bacteroidetes;c _(—) Bacteroidia; o _(—) Bacteroidales; f _(—) Bacteroidaceae; g _(—)Bacteroides; s _(—) ovatus 196139 k _(—) Bacteria; p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g_; s_(—)198184 k _(—) Bacteria; p _(—) Firmicutes; c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g_; s_(—) 198941 k _(—) Bacteria;p _(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g_; s_(—) 2331530 k _(—) Bacteria; p _(—)Bacteroidetes; c _(—) Bacteroidia; o _(—) Bacteroidales; f _(—)Bacteroidaceae; g _(—) Bacteroides; s _(—) uniformis 269611 k _(—)Bacteria; p _(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f_(—) Ruminococcaceae; g_; s_(—) 295804 k _(—) Bacteria; p _(—)Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae; g _(—) Roseburia; s_(—) 343313 k _(—) Bacteria; p _(—)Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g_; s_(—) 361702 k _(—) Bacteria; p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Ruminococcus; s_(—) 362342 k _(—) Bacteria; p _(—) Firmicutes; c _(—)Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Ruminococcus; s_(—) 362765 k _(—) Bacteria; p _(—) Firmicutes; c _(—)Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Ruminococcus; s_(—) 370099 k _(—) Bacteria; p _(—) Firmicutes; c _(—)Erysipelotrichi; o _(—) Erysipelotrichales; f _(—) Erysipelotrichaceae;g_; s_(—) 4402610 k _(—) Bacteria; p _(—) Bacteroidetes; c _(—)Bacteroidia; o _(—) Bacteroidales; f _(—) [Odoribacteraceae]; g _(—)Butyricimonas; s_(—) 509709 k _(—) Bacteria; p _(—) Firmicutes; c _(—)Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g _(—) Dorea;s_(—) 556835 k _(—) Bacteria; p _(—) Firmicutes; c _(—) Clostridia; o_(—) Clostridiales; f _(—) Veillonellaceae; g _(—) Phascolarcto-bacterium; s_(—) 574038 k _(—) Bacteria; p _(—) Firmicutes; c _(—)Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)658370 k _(—) Bacteria; p _(—) Firmicutes; c _(—) Clostridia; o _(—)Clostridiales; f _(—) Lachnospiraceae; g_; s_(—) 823634 k _(—) Bacteria;p _(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g _(—) Ruminococcus; s _(—) bromiiNew.CleanUp.Reference k _(—) Bacteria; OTU110487 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Ruminococcus; s_(—) New.CleanUp.Reference k _(—) Bacteria; OTU112566 p_(—) Bacteroidetes; c _(—) Bacteroidia; o _(—) Bacteroidales; f _(—)Bacteroidaceae; g _(—) Bacteroides; s _(—) uniformisNew.Cleanup.Reference k _(—) Bacteria; OTU122371 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU124061 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU127991 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Ruminococcus; s_(—) New.CleanUp.Reference k _(—) Bacteria; OTU1320 p_(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g _(—) aecalibacterium; s _(—) prausnitziiNew.CleanUp.Reference k _(—) Bacteria; OTU134087 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU135990 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g _(—)Coprococcus; s_(—) New.CleanUp.Reference k _(—) Bacteria; OTU141755 p_(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae New.CleanUp.Reference k _(—) Bacteria; OTU141972 p _(—)Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g _(—) Ruminococcus; s_(—) New.CleanUp.Reference k _(—)Bacteria; OTU149108 p _(—) Firmicutes; c _(—) Clostridia; o _(—)Clostridiales; f _(—) Lachnospiraceae; g _(—) Lachnospira; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU152821 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f_; g_; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU153408 p _(—) Bacteroidetes; c_(—) Bacteroidia; o _(—) Bacteroidales; f _(—) Bacteroidaceae; g _(—)Bacteroides; s _(—) uniformis New.CleanUp.Reference k _(—) Bacteria;OTU153961 p _(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f_(—) Ruminococcaceae New.CleanUp.Reference k _(—) Bacteria; OTU156924 p_(—) Bacteroidetes; c _(—) Bacteroidia; o _(—) Bacteroidales; f _(—)Bacteroidaceae; g _(—) Bacteroides; s_(—) New.CleanUp.Reference k _(—)Bacteria; OTU164203 p _(—) Firmicutes; c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g _(—) Oscillospira; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU28435 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g _(—)Coprococcus; s_(—) New.CleanUp.Reference k _(—) Bacteria; OTU37938 p_(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae; g _(—) Blautia; s_(—) New.CleanUp.Reference k _(—)Bacteria; OTU41338 p _(—) Firmicutes; c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g _(—) Ruminococcus; s _(—)bromii New.CleanUp.Reference k _(—) Bacteria; OTU41725 p _(—)Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f_; g_; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU47134 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) RuminococcaceaeNew.CleanUp.Reference k _(—) Bacteria; OTU5050 p _(—) Firmicutes; c _(—)Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g _(—)Lachnospira; s_(—) New.CleanUp.Reference k _(—) Bacteria; OTU57003 p_(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae; g _(—) Coprococcus; s_(—) New.CleanUp.Reference k _(—)Bacteria; OTU58395 p _(—) Firmicutes; c _(—) Clostridia; o _(—)Clostridiales; f _(—) Lachnospiraceae; g _(—) Coprococcus; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU58632 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Ruminococcus; s_(—) New.CleanUp.Reference k _(—) Bacteria; OTU74051 p_(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g _(—) Faecalibacterium; s _(—) prausnitziiNew.CleanUp.Reference k _(—) Bacteria; OTU79183 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU80284 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU86790 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU86928 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Faecalibacterium; s _(—) prausnitzii New.CleanUp.Reference k _(—)Bacteria; OTU89172 p _(—) Firmicutes; c _(—) Clostridia; o _(—)Clostridiales; f _(—) Lachnospiraceae; g _(—) Coprococcus; s_(—)New.CleanUp.Reference k _(—) Bacteria; OTU92834 p _(—) Firmicutes; c_(—) Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Oscillospira; s_(—) New.CleanUp.Reference k _(—) Bacteria; OTU97550 p_(—) Firmicutes; c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae; g_; s_(—) New.CleanUp.Reference k _(—) Bacteria;OTU99007 p _(—) Bacteroidetes; c _(—) Bacteroidia; o _(—) Bacteroidales;f _(—) Porphyromonadaceae; g _(—) Parabacteroides; s_(—)New.ReferenceOTU129 k _(—) Bacteria; p _(—) Bacteroidetes; c _(—)Bacteroidia; o _(—) Bacteroidales; f _(—) Bacteroidaceae; g _(—)Bacteroides; s_(—)

Aspects of the disclosure comprise determining an “OTU abundance score”.An OTU abundance score may be determined to diagnose a subject with afood allergy and/or to identify a patient as a candidate for foodallergy therapy. An OTU abundance score may be calculated from two ormore OTUs, such as the OTUs listed in Table 1. In some embodiments, anOTU abundance score is calculated using 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 ofthe OTUs listed in Table 1. In some embodiments, an OTU abundance scoreis calculated by first adding a rarefied absolute count matrix of OTUsby offset 1.0 and log 10-transforming, then scaling by dividing thevalue by their root mean square across samples. The abundance ofallergic-abundant OTUs is multiplied by (−1).

In some embodiments, the disclosed methods are for treating allergies,asthma, diabetes (e.g. type 1 diabetes), graft rejection, arthritis(rheumatoid arthritis such as acute arthritis, chronic rheumatoidarthritis, gout or gouty arthritis, acute gouty arthritis, acuteimmunological arthritis, chronic inflammatory arthritis, degenerativearthritis, type II collagen-induced arthritis, infectious arthritis,Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still'sdisease, vertebral arthritis, and systemic juvenile-onset rheumatoidarthritis, osteoarthritis, arthritis chronica progrediente, arthritisdeformans, polyarthritis chronica primaria, reactive arthritis, andankylosing spondylitis), inflammatory hyperproliferative skin diseases,psoriasis such as plaque psoriasis, gutatte psoriasis, pustularpsoriasis, and psoriasis of the nails, atopy including atopic diseasessuch as hay fever and Job's syndrome, dermatitis including contactdermatitis, chronic contact dermatitis, exfoliative dermatitis, allergicdermatitis, allergic contact dermatitis, dermatitis herpetiformis,nummular dermatitis, seborrheic dermatitis, non-specific dermatitis,primary irritant contact dermatitis, and atopic dermatitis, x-linkedhyper IgM syndrome, allergic intraocular inflammatory diseases,urticaria such as chronic allergic urticaria and chronic idiopathicurticaria, including chronic autoimmune urticaria, myositis,polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermalnecrolysis, scleroderma (including systemic scleroderma), sclerosis suchas systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS,primary progressive MS (PPMS), and relapsing remitting MS (RRMS),progressive systemic sclerosis, atherosclerosis, arteriosclerosis,sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO),inflammatory bowel disease (IBD) (for example, Crohn's disease,autoimmune-mediated gastrointestinal diseases, colitis such asulcerative colitis, colitis ulcerosa, microscopic colitis, collagenouscolitis, colitis polyposa, necrotizing enterocolitis, and transmuralcolitis, and autoimmune inflammatory bowel disease), bowel inflammation,pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis,respiratory distress syndrome, including adult or acute respiratorydistress syndrome (ARDS), meningitis, inflammation of all or part of theuvea, iritis, choroiditis, an autoimmune hematological disorder,rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema,cranial nerve damage as in meningitis, herpes gestationis, pemphigoidgestationis, pruritis scroti, autoimmune premature ovarian failure,sudden hearing loss due to an autoimmune condition, IgE-mediateddiseases such as anaphylaxis and allergic and atopic rhinitis,encephalitis such as Rasmussen's encephalitis and limbic and/orbrainstem encephalitis, uveitis, such as anterior uveitis, acuteanterior uveitis, granulomatous uveitis, nongranulomatous uveitis,phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis,glomerulonephritis (GN) with and without nephrotic syndrome such aschronic or acute glomerulonephritis such as primary GN, immune-mediatedGN, membranous GN (membranous nephropathy), idiopathic membranous GN oridiopathic membranous nephropathy, membrano- or membranous proliferativeGN (MPGN), including Type I and Type II, and rapidly progressive GN,proliferative nephritis, autoimmune polyglandular endocrine failure,balanitis including balanitis circumscripta plasmacellularis,balanoposthitis, erythema annulare centrifugum, erythema dyschromicumperstans, eythema multiform, granuloma annulare, lichen nitidus, lichensclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus,lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis,premalignant keratosis, pyoderma gangrenosum, allergic conditions andresponses, allergic reaction, eczema including allergic or atopiceczema, asteatotic eczema, dyshidrotic eczema, and vesicularpalmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma,and auto-immune asthma, conditions involving infiltration of T cells andchronic inflammatory responses, immune reactions against foreignantigens such as fetal A-B-O blood groups during pregnancy, chronicpulmonary inflammatory disease, autoimmune myocarditis, leukocyteadhesion deficiency, lupus, including lupus nephritis, lupus cerebritis,pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus anddiscoid lupus erythematosus, alopecia lupus, systemic lupuserythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE,neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus,juvenile onset (Type I) diabetes mellitus, including pediatricinsulin-dependent diabetes mellitus (IDDM), and adult onset diabetesmellitus (Type II diabetes) and autoimmune diabetes. Also contemplatedare immune responses associated with acute and delayed hypersensitivitymediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosisincluding lymphomatoid granulomatosis, Wegener's granulomatosis,agranulocytosis, vasculitides, including vasculitis, large-vesselvasculitis (including polymyalgia rheumatica and gianT cell (Takayasu's)arteritis), medium-vessel vasculitis (including Kawasaki's disease andpolyarteritis nodosa/periarteritis nodosa), microscopic polyarteritis,immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivityvasculitis, necrotizing vasculitis such as systemic necrotizingvasculitis, and ANCA-associated vasculitis, such as Churg-Straussvasculitis or syndrome (CSS) and ANCA-associated small-vesselvasculitis, temporal arteritis, aplastic anemia, autoimmune aplasticanemia, Coombs positive anemia, Diamond Blackfan anemia, hemolyticanemia or immune hemolytic anemia including autoimmune hemolytic anemia(AIHA), Addison's disease, autoimmune neutropenia, pancytopenia,leukopenia, diseases involving leukocyte diapedesis, CNS inflammatorydisorders, Alzheimer's disease, Parkinson's disease, multiple organinjury syndrome such as those secondary to septicemia, trauma orhemorrhage, antigen-antibody complex-mediated diseases, anti-glomerularbasement membrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture'ssyndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnsonsyndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid,pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigusmucus-membrane pemphigoid, and pemphigus erythematosus), autoimmunepolyendocrinopathies, Reiter's disease or syndrome, thermal injury,preeclampsia, an immune complex disorder such as immune complexnephritis, antibody-mediated nephritis, polyneuropathies, chronicneuropathy such as IgM polyneuropathies or IgM-mediated neuropathy,autoimmune or immune-mediated thrombocytopenia such as idiopathicthrombocytopenic purpura (ITP) including chronic or acute ITP, scleritissuch as idiopathic cerato-scleritis, episcleritis, autoimmune disease ofthe testis and ovary including autoimmune orchitis and oophoritis,primary hypothyroidism, hypoparathyroidism, autoimmune endocrinediseases including thyroiditis such as autoimmune thyroiditis,Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), orsubacute thyroiditis, autoimmune thyroid disease, idiopathichypothyroidism, Grave's disease, polyglandular syndromes such asautoimmune polyglandular syndromes (or polyglandular endocrinopathysyndromes), paraneoplastic syndromes, including neurologicparaneoplastic syndromes such as Lambert-Eaton myasthenic syndrome orEaton-Lambert syndrome, stiff-man or stiff-person syndrome,encephalomyelitis such as allergic encephalomyelitis orencephalomyelitis allergica and experimental allergic encephalomyelitis(EAE), experimental autoimmune encephalomyelitis, myasthenia gravis suchas thymoma-associated myasthenia gravis, cerebellar degeneration,neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), andsensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome,autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, gianT cellhepatitis, chronic active hepatitis or autoimmune chronic activehepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitisobliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger'sdisease (IgA nephropathy), idiopathic IgA nephropathy, linear IgAdermatosis, acute febrile neutrophilic dermatosis, subcorneal pustulardermatosis, transient acantholytic dermatosis, cirrhosis such as primarybiliary cirrhosis and pneumonocirrhosis, autoimmune enteropathysyndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy),refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophiclateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease,autoimmune ear disease such as autoimmune inner ear disease (AIED),autoimmune hearing loss, polychondritis such as refractory or relapsedor relapsing polychondritis, pulmonary alveolar proteinosis, Cogan'ssyndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet'sdisease/syndrome, rosacea autoimmune, zoster-associated pain,amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis,which includes monoclonal B cell lymphocytosis (e.g., benign monoclonalgammopathy and monoclonal gammopathy of undetermined significance,MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathiessuch as epilepsy, migraine, arrhythmia, muscular disorders, deafness,blindness, periodic paralysis, and channelopathies of the CNS, autism,inflammatory myopathy, focal or segmental or focal segmentalglomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis,chorioretinitis, autoimmune hepatological disorder, fibromyalgia,multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastricatrophy, presenile dementia, demyelinating diseases such as autoimmunedemyelinating diseases and chronic inflammatory demyelinatingpolyneuropathy, Dressler's syndrome, alopecia greata, alopecia totalis,CREST syndrome (calcinosis, Raynaud's phenomenon, esophagealdysmotility, sclerodactyl), and telangiectasia), male and femaleautoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixedconnective tissue disease, Chagas' disease, rheumatic fever, recurrentabortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome,Cushing's syndrome, bird-fancier's lung, allergic granulomatousangiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitissuch as allergic alveolitis and fibrosing alveolitis, interstitial lungdisease, transfusion reaction, leprosy, malaria, parasitic diseases suchas leishmaniasis, kypanosomiasis, schistosomiasis, ascariasis,aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue,endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonaryfibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathicpulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatumet diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, flariasis, cyclitis such as chroniccyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), orFuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus(HIV) infection, SCID, acquired immune deficiency syndrome (AIDS),echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis,parvovirus infection, rubella virus infection, post-vaccinationsyndromes, congenital rubella infection, Epstein-Barr virus infection,mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea,post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis,tabes dorsalis, chorioiditis, gianT cell polymyalgia, chronichypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemickeratoconjunctivitis, idiopathic nephritic syndrome, minimal changenephropathy, benign familial and ischemia-reperfusion injury, transplantorgan reperfusion, retinal autoimmunity, joint inflammation, bronchitis,chronic obstructive airway/pulmonary disease, silicosis, aphthae,aphthous stomatitis, arteriosclerotic disorders, asperniogenese,autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren'scontracture, endophthalmia phacoanaphylactica, enteritis allergica,erythema nodosum leprosum, idiopathic facial paralysis, chronic fatiguesyndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearingloss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis,leucopenia, mononucleosis infectiosa, traverse myelitis, primaryidiopathic myxedema, nephrosis, ophthalmia symphatica, orchitisgranulomatosa, pancreatitis, polyradiculitis acuta, pyodermagangrenosum, Quervain's thyreoiditis, acquired spenic atrophy,non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning,conditions involving infiltration of T cells, leukocyte-adhesiondeficiency, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, diseasesinvolving leukocyte diapedesis, multiple organ injury syndrome,antigen-antibody complex-mediated diseases, antiglomerular basementmembrane disease, allergic neuritis, autoimmune polyendocrinopathies,oophoritis, primary myxedema, autoimmune atrophic gastritis, sympatheticophthalmia, rheumatic diseases, mixed connective tissue disease,nephrotic syndrome, insulitis, polyendocrine failure, autoimmunepolyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism(AOIH), cardiomyopathy such as dilated cardiomyopathy, epidermolisisbullosa acquisita (EBA), hemochromatosis, myocarditis, nephroticsyndrome, primary sclerosing cholangitis, purulent or nonpurulentsinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, orsphenoid sinusitis, an eosinophil-related disorder such as eosinophilia,pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome,Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonaryeosinophilia, bronchopneumonic aspergillosis, aspergilloma, orgranulomas containing eosinophils, anaphylaxis, seronegativespondyloarthritides, polyendocrine autoimmune disease, sclerosingcholangitis, sclera, episclera, chronic mucocutaneous candidiasis,Bruton's syndrome, transient hypogammaglobulinemia of infancy,Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis,autoimmune disorders associated with collagen disease, rheumatism,neurological disease, lymphadenitis, reduction in blood pressureresponse, vascular dysfunction, tissue injury, cardiovascular ischemia,hyperalgesia, renal ischemia, cerebral ischemia, and diseaseaccompanying vascularization, allergic hypersensitivity disorders,glomerulonephritides, reperfusion injury, ischemic re-perfusiondisorder, reperfusion injury of myocardial or other tissues,lymphomatous tracheobronchitis, inflammatory dermatoses, dermatoses withacute inflammatory components, multiple organ failure, bullous diseases,renal cortical necrosis, acute purulent meningitis or other centralnervous system inflammatory disorders, ocular and orbital inflammatorydisorders, granulocyte transfusion-associated syndromes,cytokine-induced toxicity, narcolepsy, acute serious inflammation,chronic intractable inflammation, pyelitis, endarterial hyperplasia,peptic ulcer, valvulitis, graft versus host disease, contacthypersensitivity, asthmatic airway hyperreaction, or endometriosis.

V. Kits

Certain aspects of the present disclosure also concern kits containingcompositions of the disclosure or compositions to implement methods ofthe disclosure. In some embodiments, kits can be used to evaluate one ormore biomarkers. In certain embodiments, a kit contains, contains atleast or contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,100, 500, 1,000 or more probes, primers or primer sets, syntheticmolecules or inhibitors, or any value or range and combination derivabletherein. In some embodiments, there are kits for evaluating biomarkeractivity in a cell.

Kits may comprise components, which may be individually packaged orplaced in a container, such as a tube, bottle, vial, syringe, or othersuitable container means.

Individual components may also be provided in a kit in concentratedamounts; in some embodiments, a component is provided individually inthe same concentration as it would be in a solution with othercomponents. Concentrations of components may be provided as 1×, 2×, 5×,10×, or 20× or more.

Kits for using probes, synthetic nucleic acids, non-synthetic nucleicacids, and/or inhibitors of the disclosure for prognostic or diagnosticapplications are included as part of the disclosure. Specificallycontemplated are any such molecules corresponding to any biomarkeridentified herein, which includes nucleic acid primers/primer sets andprobes that are identical to or complementary to all or part of abiomarker, which may include noncoding sequences of the biomarker, aswell as coding sequences of the biomarker.

In certain aspects, negative and/or positive control nucleic acids,probes, and inhibitors are included in some kit embodiments. Inaddition, a kit may include a sample that is a negative or positivecontrol for one or more biomarkers.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein and that different embodiments may be combined. The claimsoriginally filed are contemplated to cover claims that are multiplydependent on any filed claim or combination of filed claims.

Any embodiment of the disclosure involving specific biomarker by name iscontemplated also to cover embodiments involving biomarkers whosesequences are at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99% identical to the mature sequence of thespecified nucleic acid.

Embodiments of the disclosure include kits for analysis of apathological sample by assessing biomarker profile for a samplecomprising, in suitable container means, two or more biomarker probes,wherein the biomarker probes detect one or more of the biomarkersidentified herein. The kit can further comprise reagents for labelingnucleic acids in the sample. The kit may also include labeling reagents,including at least one of amine-modified nucleotide, poly(A) polymerase,and poly(A) polymerase buffer. Labeling reagents can include anamine-reactive dye.

Examples

The following examples are included to demonstrate embodiments of theinvention. It should be appreciated by those of skill in the art thatthe techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute certain modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1—Identification of a Protective Fetal Microbial Profile inHealthy Twins

Methods

Study Design

A design of the analytical workflow is shown in FIG. 1 , whichillustrates enrollment of the twin pairs that were food allergic andthose that were healthy. Statistical tests used Wilcoxon rank-sum testsfor analysis of data.

Human Fecal Sample Collection

Participants were recruited as part of an observational study(ClinicalTrials.gov Identifier: NCT01613885) at multiple sites from 2014to 2018. Food allergic participants in this study were diagnosed withfood allergy by a staged and validated food challenge (13) performed bytrained center staff. Fecal samples were collected per a standardoperating procedure developed by the NIH Microbiome Project (21). Fecalsamples were collected from 18 pairs of twins. Among 18 twin pairs, 13were discordant for food allergy (one sibling had food allergy and theother was healthy) and 5 were concordant for food allergy (both siblingswere food allergy). No one was on any medications or experienced anyrespiratory infections (e.g. cold, flu, pneumonia) at the time of fecalsample collection. All samples that passed QC (n=34 for microbiotaanalysis; n=36 for metabolite analysis) were used for statisticalcomparisons.

16S rRNA-Targeted Library Preparation and Sequencing

Bacterial DNA was extracted from fecal samples of the twin cohort usingthe Power Soil DNA Isolation Kit (MoBio). 16S rRNA-targeted geneamplicon library preparation and sequencing were performed at theEnvironmental Sample Preparation and Sequencing Facility at ArgonneNational Laboratory (DuPage, Illinois). The V4 region of the 16S rRNAgene was amplified by PCR with region-specific primers (515F-806R) thatinclude sequencer adapter sequences used in the Illumina flowcell. 151bp paired-end (PE) reads with 12 bp barcodes were generated followingpreviously described protocols (22) on an Illumina MiSegm instrument. Onaverage, each sample yielded 183,952±7,011 (mean±S.E.M.; ranging from94,917 to 268,423) read pairs. One sample (S5077, allergic sibling of atwin pair) failed sequencing and yielded 0 reads and the correspondingtwin pair (#13) was therefore excluded from 16S data analysis. A totalof 34 samples (from 22 allergic and 12 healthy twins) was kept forfurther analysis, including 12 discordant twin pairs (n=24) and 5concordant twin pairs (n=10).

16S rRNA Microbial Quantification and Normalization

The microbial 16S rRNA-targeted gene amplicon sequencing data wereprocessed by Quantitative Insights into Microbial Ecology (QIIME)(version 1.9) (23) using a procedure similar to what has been previouslydescribed (24). In brief, low-quality bases were trimmed at 5′ end ofraw PE reads and 3′ overlapping mates were merged by SeqPrep (Availableon the World Wide Web at github.com/jstjohn/SeqPrep). The open referenceOTU picking protocol was used at 97% sequence identity against theGreengenes database (August 2013 release) (25). Reads were aligned toreference sequences using PyNAST (26) and taxonomy was assigned usinguclust consensus taxonomy assigner (27). Chimeric sequences wereidentified and removed using ChimeraSlayer (v20110519) (28). Sequenceswith “Unassigned” taxa at the kingdom level were also removed. Data werethen rarefied to an even depth of 92,670 sequences across all samples(n=3734 the twin cohort). Alpha-diversity (Shannon index) was comparedbetween the allergic and healthy groups using Wilcoxon rank-sum test(unpaired, non-parametric) for all samples or Wilcoxon signed-rank test(paired, non-parametric) within the discordant twin pairs only.Beta-diversity metrics were calculated and compared between the twogroups using PERMANOVA with weighted UniFrac distance in R package vegan(v2.4.5) (29).

Differentially Abundant Microbial Taxa Identification

Bacterial taxa differentially abundant between allergic and healthygroups of the twin cohort were identified using the following approach.First, OTUs present in fewer than 4 samples were removed. Second, forall samples (n=34), relative abundance of OTUs was compared between thetwo groups using Discrete False-Discovery Rate (DS-FDR) (30) method(hereafter referred to as test #1) with parameters “transformtype=normdata, method=meandiff, alpha=0.10, numperm=1000,fdr_method=dsfdr” (accessed 10102018) (available on the World Wide Webat github.com/biocore/dsFDR). The DS-FDR algorithm computes teststatistics, raw P-values, and estimates false-discovery rate from apermutation test (default 1000 permutations). Of 5,590 OTUs total, 180reached P<0.05; none reached FDR 0.10 potentially due to sample size.Then, within the discordant twin pairs (n=24, from 12 pairs), for whichone sibling is allergic and the other is healthy, relative abundance ofOTUs was compared between groups using Wilcoxon signed-rank test(paired, non-parametric) (hereafter referred to as test #2). Of 5,590OTUs total, 259 reached a significance level of P<0.10. A more lenientP-value cutoff (0.10) was used here considering that non-parametricrank-based method has less power than DS-FDR method (30). AfterBenjamini-Hochberg FDR (BH-FDR) correction (31) for multiple testing, noOTUs passed FDR 0.10 potentially due to small sample sizes. Between 180OTUs returned by test #1 and 259 OTUs returned by test #2, 64 OTUsoverlapped and showed consistent direction of change in abundance (moreabundant in healthy or allergic) in both tests and were kept for furtheranalysis (OTUs shown in Table 2).

TABLE 2 List of 64 OTUs identified herein as differentially abundantbetween healthy and allergic twins. OTU Taxonomy Direction OTU Type173135 k _(—) Bacteria; UP in Healthy- p _(—) Firmicutes; Healthyabundant c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g_; s_(—) 174588 k _(—) Bacteria; UP in Healthy- p _(—)Firmicutes; Healthy abundant c _(—) Clostridia; o _(—) Clostridiales; f_(—) Lachnospiraceae; g_; s_(—) 174818 k _(—) Bacteria; UP in Healthy- p_(—) Firmicutes; Healthy abundant c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g_; s_(—) 176077 k _(—) Bacteria;UP in Healthy- p _(—) Firmicutes; Healthy abundant c _(—) Clostridia; o_(—) Clostridiales; f_; g_; s_(—) 176664 k _(—) Bacteria; UP in Healthy-p _(—) Firmicutes; Healthy abundant c _(—) Clostridia; o _(—)Clostridiales; f _(—) Lachnospiraceae; g _(—) Roseburia; s _(—) faecis178799 k _(—) Bacteria; UP in Healthy- p _(—) Firmicutes; Healthyabundant c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g_; s_(—) 186478 k _(—) Bacteria; UP in Healthy- p _(—)Firmicutes; Healthy abundant c _(—) Clostridia; o _(—) Clostridiales; f_(—) Ruminococcaceae; g_; s_(—) 188079 k _(—) Bacteria; UP in Healthy- p_(—) Firmicutes; Healthy abundant c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g _(—) Ruminococcus 189816 k _(—)Bacteria; UP in Allergic- p _(—) Firmicutes; Allergic Abundant c _(—)Clostridia; o _(—) Clostridiales 190169 k _(—) Bacteria; UP in Healthy-p _(—) Firmicutes; Healthy abundant c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g_; s_(—) 190649 k _(—) Bacteria;UP in Healthy- p _(—) Firmicutes; Healthy abundant c _(—) Clostridia; o_(—) Clostridiales; f _(—) Ruminococcaceae; g_; s_(—) 195258 k _(—)Bacteria; UP in Healthy- p _(—) Bacteroidetes; Healthy abundant c _(—)Bacteroidia; o _(—) Bacteroidales; f _(—) Bacteroidaceae; g _(—)Bacteroides; s _(—) ovatus 196139 k _(—) Bacteria; UP in Healthy- p _(—)Firmicutes; Healthy abundant c _(—) Clostridia; o _(—) Clostridiales; f_(—) Ruminococcaceae; g_; s_(—) 198184 k _(—) Bacteria; UP in Healthy- p_(—) Firmicutes; Healthy abundant c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g_; s_(—) 198941 k _(—) Bacteria;UP in Healthy- p _(—) Firmicutes; Healthy abundant c _(—) Clostridia; o_(—) Clostridiales; f _(—) Ruminococcaceae; g_; s_(—) 2331530 k _(—)Bacteria; UP in Healthy- p _(—) Bacteroidetes; Healthy abundant c _(—)Bacteroidia; o _(—) Bacteroidales; f _(—) Bacteroidaceae; g _(—)Bacteroides; s _(—) uniformis 269611 k _(—) Bacteria; UP in Healthy- p_(—) Firmicutes; Healthy abundant c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g_; s_(—) 295804 k _(—) Bacteria;UP in Healthy- p _(—) Firmicutes; Healthy abundant c _(—) Clostridia; o_(—) Clostridiales; f _(—) Lachnospiraceae; g _(—) Roseburia; s_(—)343313 k _(—) Bacteria; UP in Healthy- p _(—) Firmicutes; Healthyabundant c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g_; s_(—) 361702 k _(—) Bacteria; UP in Healthy- p _(—)Firmicutes; Healthy abundant c _(—) Clostridia; o _(—) Clostridiales; f_(—) Ruminococcaceae; g _(—) Ruminococcus; s_(—) 362342 k _(—) Bacteria;UP in Healthy- p _(—) Firmicutes; Healthy abundant c _(—) Clostridia; o_(—) Clostridiales; f _(—) Ruminococcaceae; g _(—) Ruminococcus; s_(—)362765 k _(—) Bacteria; UP in Healthy- p _(—) Firmicutes; Healthyabundant c _(—) Clostridia; o _(—) Clostridiales; f _(—)Ruminococcaceae; g _(—) Ruminococcus; s_(—) 370099 k _(—) Bacteria; UPin Healthy- p _(—) Firmicutes; Healthy abundant c _(—) Erysipelotrichi;o _(—) Erysipelotrichales; f _(—) Erysipelotrichaceae; g_; s_(—) 4402610k _(—) Bacteria; UP in Healthy- p _(—) Bacteroidetes; Healthy abundant c_(—) Bacteroidia; o _(—) Bacteroidales; f _(—) [Odoribacteraceae]; g_(—) Butyricimonas; s_(—) 509709 k _(—) Bacteria; UP in Healthy- p _(—)Firmicutes; Healthy abundant c _(—) Clostridia; o _(—) Clostridiales; f_(—) Lachnospiraceae; g _(—) Dorea; s_(—) 556835 k _(—) Bacteria; UP inHealthy- p _(—) Firmicutes; Healthy abundant c _(—) Clostridia; o _(—)Clostridiales; f _(—) Veillonellaceae; g _(—) Phascolarcto- bacterium;s_(—) 574038 k _(—) Bacteria; UP in Healthy- p _(—) Firmicutes; Healthyabundant c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae; g_; s_(—) 658370 k _(—) Bacteria; UP in Healthy- p _(—)Firmicutes; Healthy abundant c _(—) Clostridia; o _(—) Clostridiales; f_(—) Lachnospiraceae; g_; s_(—) 823634 k _(—) Bacteria; UP in Healthy- p_(—) Firmicutes; Healthy abundant c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g _(—) Ruminococcus; s _(—)bromii New.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p _(—)Firmicutes; Healthy abundant OTU110487 c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g _(—) Ruminococcus; s_(—)New.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p _(—)Bacteroidetes; Healthy abundant OTU112566 c _(—) Bacteroidia; o _(—)Bacteroidales; f _(—) Bacteroidaceae; g _(—) Bacteroides; s _(—)uniformis New.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p _(—)Firmicutes; Healthy abundant OTU122371 c _(—) Clostridia; o _(—)Clostridiales; f _(—) Lachnospiraceae; g_; s_(—) New.CleanUp. k _(—)Bacteria; UP in Healthy- Reference p _(—) Firmicutes; Healthy abundantOTU124061 c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae; g_; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU127991 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Ruminococcus; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU1320 c _(—) Clostridia;o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—) Faecalibacterium; s_(—) prausnitzii New.CleanUp. k _(—) Bacteria; UP in Healthy- Referencep _(—) Firmicutes; Healthy abundant OTU134087 c _(—) Clostridia; o _(—)Clostridiales; f _(—) Lachnospiraceae; g_; s_(—) New.CleanUp. k _(—)Bacteria; UP in Healthy- Reference p _(—) Firmicutes; Healthy abundantOTU135990 c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae; g _(—) Coprococcus; s_(—) New.CleanUp. k _(—) Bacteria;UP in Healthy- Reference p _(—) Firmicutes; Healthy abundant OTU141755 c_(—) Clostridia; o _(—) Clostridiales; f _(—) LachnospiraceaeNew.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p _(—)Firmicutes; Healthy abundant OTU141972 c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g _(—) Ruminococcus; s_(—)New.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p _(—)Firmicutes; Healthy abundant OTU149108 c _(—) Clostridia; o _(—)Clostridiales; f _(—) Lachnospiraceae; g _(—) Lachnospira; s_(—)New.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p _(—)Firmicutes; Healthy abundant OTU152821 c _(—) Clostridia; o _(—)Clostridiales; f_; g_; s_(—) New.CleanUp. k _(—) Bacteria; UP inHealthy- Reference p _(—) Bacteroidetes; Healthy abundant OTU153408 c_(—) Bacteroidia; o _(—) Bacteroidales; f _(—) Bacteroidaceae; g _(—)Bacteroides; s _(—) uniformis New.CleanUp. k _(—) Bacteria; UP inHealthy- Reference p _(—) Firmicutes; Healthy abundant OTU153961 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae New.CleanUp. k_(—) Bacteria; UP in Healthy- Reference p _(—) Bacteroidetes; Healthyabundant OTU156924 c _(—) Bacteroidia; o _(—) Bacteroidales; f _(—)Bacteroidaceae; g _(—) Bacteroides; s_(—) New.CleanUp. k _(—) Bacteria;UP in Healthy- Reference p _(—) Firmicutes; Healthy abundant OTU164203 c_(—) Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Oscillospira; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU28435 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g _(—)Coprococcus; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU37938 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g _(—)Blautia; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p_(—) Firmicutes; Healthy abundant OTU41338 c _(—) Clostridia; o _(—)Clostridiales; f _(—) Ruminococcaceae; g _(—) Ruminococcus; s _(—)bromii New.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p _(—)Firmicutes; Healthy abundant OTU41725 c _(—) Clostridia; o _(—)Clostridiales; f_; g_; s_(—) New.CleanUp. k _(—) Bacteria; UP inHealthy- Reference p _(—) Firmicutes; Healthy abundant OTU47134 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae New.CleanUp. k_(—) Bacteria; UP in Healthy- Reference p _(—) Firmicutes; Healthyabundant OTU5050 c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae; g _(—) Lachnospira; s_(—) New.CleanUp. k _(—) Bacteria;UP in Healthy- Reference p _(—) Firmicutes; Healthy abundant OTU57003 c_(—) Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g _(—)Coprococcus; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU58395 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g _(—)Coprococcus; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU58632 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Ruminococcus; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU74051 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Faecalibacterium; s _(—) prausnitzii New.CleanUp. k _(—) Bacteria; UP inHealthy- Reference p _(—) Firmicutes; Healthy abundant OTU79183 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)New.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p _(—)Firmicutes; Healthy abundant OTU80284 c _(—) Clostridia; o _(—)Clostridiales; f _(—) Lachnospiraceae; g_; s_(—) New.CleanUp. k _(—)Bacteria; UP in Allergic- Reference p _(—) Firmicutes; Allergic AbundantOTU86790 c _(—) Clostridia; o _(—) Clostridiales; f _(—)Lachnospiraceae; g_; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU86928 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Faecalibacterium; s _(—) prausnitzii New.CleanUp. k _(—) Bacteria; UP inHealthy- Reference p _(—) Firmicutes; Healthy abundant OTU89172 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g _(—)Coprococcus; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU92834 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Ruminococcaceae; g _(—)Oscillospira; s_(—) New.CleanUp. k _(—) Bacteria; UP in Healthy-Reference p _(—) Firmicutes; Healthy abundant OTU97550 c _(—)Clostridia; o _(—) Clostridiales; f _(—) Lachnospiraceae; g_; s_(—)New.CleanUp. k _(—) Bacteria; UP in Healthy- Reference p _(—)Bacteroidetes; Healthy abundant OTU99007 c _(—) Bacteroidia; o _(—)Bacteroidales; f _(—) Porphyromonadaceae; g _(—) Parabacteroides; s_(—)New.Reference k _(—) Bacteria; UP in Healthy- OTU129 p _(—)Bacteroidetes; Healthy abundant c _(—) Bacteroidia; o _(—)Bacteroidales; f _(—) Bacteroidaceae; g _(—) Bacteroides; s_(—)

OTU Abundance Score Calculation

Out of 64 OTUs differentially abundant between allergic and healthy twinmembers, 62 were more abundant in the healthy group (hereby referred toas healthy-abundant OTUs), and 2 were more abundant in the allergicgroup (hereby referred to as allergic-abundant OTUs) (see Table 2 andFIGS. 5, 6, and 7A). The limited number of allergic-abundant OTUs didnot warrant the calculation of an OTU ratio as previously made (23),defined as the total number of potentially healthy-abundant OTUs dividedby the total number of potentially allergic-abundant OTUs per sample.Here, an “OTU abundance score” was calculated as an aggregated signaturetaking into consideration the relative abundance of the 64 OTUs shown inTable 2 and FIG. 6 . First, the rarefied absolute count matrix of OTUswas added by offset 1.0 and log 10-transformed to bring data to close toGaussian distribution, then scaled by dividing the value by their rootmean square across samples. The abundance of allergic-abundant OTUs wasmultiplied by (−1). Second, the sum of the transformed abundance of the64 OTUs was calculated to generate the aggregate score.

Results

Microbial signatures and metabolomic profiles were examined in the fecalsamples from a unique collection of twin pairs that were raised in thesame household in which they equally avoided the foods that the affectedtwin was found to be allergic to and were either concordant ordiscordant for food allergy (FIG. 2 ). Baseline demographic and clinicalcharacteristics of the twin cohort are shown in Tables 3-1 and 3-2. Theaverage age of participants at sample collection was 39.4±4.1 years(mean±SEM). All of the twins lived independently after the age of 19years. There were no significant differences in the baselinedemographical and clinical characteristics between the healthy vs.food-allergic twin pairs.

TABLE 3-1 Baseline demographic and clinical characteristics of the twincohort Demographics Breast- Discordant Age at Body feeding for TwinSample or stool Mass at least 6 pairs ID# concordant Zygosity collectionSex Race Index mo 1 S0003 concordant Monozygotic 57 Male White 25.77 noS0004 concordant Monozygotic 57 Male White 26.70 no 2 S0005 discordantMonozygotic 0.5 Female Asian and 18.23 Yes White S0006 discordantMonozygotic 0.5 Female Asian and 18.95 Yes White 3 S0008 discordantDizygotic 6 Male Asian 12.63 Yes S0007 discordant Dizygotic 6 FemaleAsian NA Yes 4 S0017 concordant Monozygotic 1 Female White 17.55 YesS0018 concordant Monozygotic 1 Female White 16.82 Yes 5 S0021 discordantMonozygotic 5 Male Asian 15.09 No S0022 discordant Monozygotic 5 MaleAsian 15.08 No 6 S0023 discordant Dizygotic 5 Female White 13.10 YesS0024 discordant Dizygotic 5 Female White 13.75 Yes 7 S1376 discordantMonozygotic 58 Male Hispanic 27.62 Yes or Latino S1375 discordantMonozygotic 58 Male Hispanic 26.87 Yes or Latino 8 S1610 discordantMonozygotic 48 Female Asian 27.79 No S1609 discordant Monozygotic 48Female Asian 26.63 No 9 S1899 discordant Dizygotic 57 Female White 29.35Yes S1900 discordant Dizygotic 57 Male White 31.37 Yes 10 S2028discordant Monozygotic 53 Female White 22.25 No S2027 discordantMonozygotic 53 Female White 21.57 No 11 S2378 discordant Monozygotic 46Female White 30.89 Yes S2377 discordant Monozygotic 46 Female White28.49 Yes 12 S2617 discordant Monozygotic 70 Female White 25.97 YesS2618 discordant Monozygotic 70 Female White 28.59 Yes 13 S5078discordant Dizygotic 47 Female Asian 31.93 Yes S5077 discordantDizygotic 47 Female Asian 25.97 Yes 14 S5765 discordant Monozygotic 57Male White 35.70 Yes S5766 discordant Monozygotic 57 Male White 34.44Yes 15 SW0011A concordant Monozygotic 58 Male White 34.35 No SW0011Bconcordant Monozygotic 58 Male White 36.12 No 16 SW0018A concordantMonozygotic 56 Female Black or 35.76 No African American SW0018Bconcordant Monozygotic 56 Female Black or 35.18 No African American 17SW0045A concordant Monozygotic 63 Male White 37.95 No SW0045B concordantMonozygotic 63 Male White 41.09 No 18 SW0057B discordant Monozygotic 22Female White 18.13 Yes SW0057A discordant Monozygotic 22 Female White19.64 Yes

TABLE 3-2 Baseline demographic and clinical characteristics of the twincohort Has a doctor Has a Food Allergy History ever told doctor Yearsyou that ever told Years with with other you have you that Twin SampleFood the peanut food atopic dermatitis you have pairs ID# allergies FoodAllergens allergy allergy or eczema? asthma? 1 S0003 Allergic Peanut,Tree nuts, 56 56 No Yes Shellfish, Soy S0004 Allergic Egg 0 55 No No 2S0005 Healthy None 0 0 Yes No S0006 Allergic Egg 0 0.3 Yes No 3 S0008Healthy None 0 0 No No S0007 Allergic Peanut, Tree nuts 5.3 5 No No 4S0017 Allergic Egg 0 0.4 No No S0018 Allergic Egg 0 0.4 No No 5 S0021Healthy None 0 0 No No S0022 Allergic Peanut, Tree nuts, 4 4 No YesFish, Mustard 6 S0023 Healthy None 0 0 No No S0024 Allergic Peanut, Egg2 2 No Yes 7 S1376 Healthy None 0 0 No Yes S1375 Allergic Oats, olives,com 0 23 No No 8 S1610 Healthy None 0 0 Yes Yes S1609 Allergic Rice,Pineapple 0 25 No Yes 9 S1899 Healthy None 0 0 No Yes S1900 AllergicPeanut, Milk 55 56 No Yes 10 S2028 Healthy None 0 0 No Yes S2027Allergic Peanut, Garbanzo 50 45 Yes Yes beans 11 S2378 Healthy None 0 0Yes No S2377 Allergic Milk, Wheat 0 45 Yes Yes 12 S2617 Healthy None 0 0No No S2618 Allergic Milk 0 68 No Yes 13 S5078 Healthy None 0 0 No NoS5077 Allergic Milk 0 46 No No 14 S5765 Healthy None 0 0 No No S5766Allergic Tree nuts 0 50 No Yes 15 SW0011A Allergic Shellfish 0 52 No YesSW0011B Allergic Milk 0 50 Yes Yes Tree nuts, Bananas, Kiwi, 16 SW0018AAllergic Avocado, Mangos, 0 50 No Yes Melons, Carrots, Cucumber SW0018BAllergic Shellfish 0 45 No Yes 17 SW0045A Allergic Apples 0 55 No YesSW0045B Allergic Tree nuts, 0 56 No No Shellfish, melons, squash 18SW0057B Healthy None 0 0 No Yes SW0057A Allergic Shellfish, Banana, 0 18No Yes crab

16S rRNA-gene amplicon sequencing was performed on fecal samples fromthe 13 healthy and 23 food-allergic age-matched twin pairs. Afterexcluding 1 sample with low sequencing depth and the corresponding twinpair, 34 samples were included for analysis, including 24 samples from12 discordant twin pairs (1 allergic, 1 healthy) and 10 samples from 5from concordant twin pairs (both allergic). An overview of theanalytical workflow is shown in FIG. 1 . The composition of commensalmicrobiota is shown in FIG. 3A. While there was sample-to-samplevariation, the presence of major families such as Bacteroidaceae,Lachnospiraceae, and Ruminococcaceae was consistent with those reportedin previous studies on fecal samples (32). For each twin pair, therelative abundance of Operational Taxonomic Units (OTUs) was compared.OTUs represent groups of microbes between closely related individuals.No significant differences were found in within-pair, sibling-wise OTUcorrelation between discordant and concordant twin pairs, or betweendizygotic and monozygotic twin pairs (FIGS. 3B and 3C). Across allsamples or within discordant twin pairs only, no significant differenceswere detected in the a-diversity (Shannon Diversity; FIGS. 3D and 3E).P-diversity (weighted UniFrac distance) metrics also did not differbetween allergic and healthy groups (FIG. 4 ).

Next the microbial composition between allergic and healthy twin pairswas compared and 64 OTUs were identified as differentially abundantbetween the two groups, with 62 OTUs higher in healthy twins (hereafterreferred to as “healthy-abundant” OTUs), and 2 OTUs higher in allergictwins (hereafter referred to as “allergic-abundant” OTUs); this is shownin FIG. 6 and in the binary presence/absence heatmap in FIG. 5 . The 62healthy-abundant OTUs were more abundant in the healthy twins comparedwith their allergic siblings, and the 2 allergic-abundant OTUs were moreabundant in the allergic twins than their healthy siblings (FIG. 7A).84% of the healthy-abundant OTUs were families in the Clostridia class,and annotated as Lachnospiraceae (n=21), Ruminococcaceae (n=28) orunclassified Clostridiales (n=4) (FIGS. 5, 6, and 7A). To develop anaggregated microbiome signature, a microbiota abundance score wascalculated taking into consideration the relative abundance of the 64differentially abundant OTUs and their change in direction betweengroups (see Methods). The OTU abundance score was significantly higherin healthy relative to allergic twins across all samples (P<0.00001)(FIG. 7B) or within discordant twins only (P=0.00049) (FIG. 8 ), asexpected, since the score was calculated from preselected OTUs. Varianceexists in the relative abundance scores for the discordant twin pairs(FIG. 8 ) because the majority of the differentially abundant OTUs arepresent in the healthy twins and absent in the allergic twins. Ifstatistical comparisons are restricted to monozygotic twins only (14pairs, 28 samples), the test statistics for the 62 healthy-abundant OTUsand the 2 allergic-abundant OTUs correlated with that of all twins (17pairs, 34 samples) (FIG. 9 ), and the OTU abundance scores remainedsignificantly different between the healthy and allergic twin groups(FIGS. 10A and 10B).

Example 2—Identification of Differential Enrichment of Fetal MetabolicPathways Between Healthy and Allergic Twins

Methods

Metabolic Profiling Sample Preparation

The metabolic profiling of human fecal samples was performed byMetabolon Inc. (Morrisville, N.C., USA). All samples were maintained at−80° C. until processed. Samples were prepared using the automatedMicroLab STAR® system from Hamilton Company (Reno, Nev., USA). Recoverystandards were added prior to the first step in the extraction processfor quality control purposes. To remove protein, to dissociate smallmolecules bound to protein or trapped in the precipitated proteinmatrix, and to recover chemically diverse metabolites, proteins wereprecipitated with methanol under vigorous shaking for 2 min (Glen MillsGenoGrinder 2000) followed by centrifugation. The resulting extract wasdivided into five fractions: two for analysis by two separate reversephase (RP)/UPLC-MS/MS methods with positive ion mode electrosprayionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ionmode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion modeESI, and one sample reserved for backup. Samples were placed briefly ona TurboVap® (Zymark) to remove the organic solvent. The sample extractswere stored overnight under nitrogen before preparation for analysis.

Metabolic Profiling Sample QA/QC

Three types of controls were analyzed together with the experimentalsamples: (1) a pooled matrix sample generated by taking a small volumeof each experimental sample as a technical replicate throughout the dataset, (2) extracted water samples as process blanks, and (3) a cocktailof QC standards carefully chosen not to interfere with the measurementof endogenous compounds were spiked into every analyzed sample, allowingfor monitoring of instrument performance and facilitatingchromatographic alignment. Instrument variability was determined bycalculating the median relative standard deviation (RSD) for thestandards that were added to each sample prior to injection into themass spectrometers (3% median RSD in this study). Overall processvariability was determined by calculating the median RSD for allendogenous metabolites (i.e., non-instrument standards) present in 100%of the pooled matrix samples (7% median RSD in this study). All 36 fecalsamples passed QC and were included in the metabolic data analysis.

Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy(UPLC-MS/MS)

All methods utilized a Waters ACQUITY ultra-performance liquidchromatography (UPLC) and a Thermo Scientific Q-Exactivehigh-resolution/accurate mass spectrometer interfaced with a heatedelectrospray ionization (HESI-II) source and Orbitrap mass analyzeroperated at 35,000 mass resolution. The sample extract was dried andreconstituted in solvents compatible to each of the four methods. Eachreconstitution solvent contained a series of standards at fixedconcentrations to ensure injection and chromatographic consistency. Onealiquot was analyzed using acidic positive ion conditions,chromatographically optimized for more hydrophilic compounds. In thismethod, the extract was gradient eluted from a C18 column (Waters UPLCBEH C18-2.1×100 mm, 1.7 μm) using water and methanol, containing 0.05%perfluoropentanoic acid (PFPA) and 0.1% formic acid (FA). Anotheraliquot was also analyzed using acidic positive ion conditions; however,it was chromatographically optimized for more hydrophobic compounds. Inthis method, the extract was gradient eluted from the same previouslymentioned C18 column using methanol, acetonitrile, water, 0.05% PFPA,and 0.01% FA, and was operated at an overall higher organic content.Another aliquot was analyzed using basic negative ion optimizedconditions using a separate dedicated C18 column. The basic extractswere gradient eluted from the column using methanol and water, howeverwith 6.5 mM ammonium bicarbonate at pH 8. The fourth aliquot wasanalyzed via negative ionization following elution from a HILIC column(Waters UPLC BEH Amide 2.1×150 mm, 1.7 μm) using a gradient consistingof water and acetonitrile with 10 mM ammonium formate, pH 10.8. The MSanalysis alternated between MS and data-dependent MSn scans usingdynamic exclusion. The scan range varied slighted between methods butcovered 70-1000 m/z.

Compound Identification and Curation

UPLC MS/MS raw data were extracted, peak-identified, and QC-processed byMetabolon Inc. (Morrisville, N.C., USA). Compounds were identified bycomparing them to internal library entries of purified standards orrecurrent unknown entities. The library was based on authenticatedstandards that contain the retention time/index (RI), mass-to-chargeratio (m/z), and chromatographic data (including MS/MS spectral data) onall molecules present in the library. Furthermore, biochemicalidentifications were based on three criteria: (1) retention index withina narrow RI window of the proposed identification, (2) accurate massmatch to the library±10 ppm, and (3) the MS/MS forward and reversescores between the experimental data and authentic standards. The MS/MSscores were based on comparing the ions in the experimental spectrum tothe ions in the library spectrum. While there may be similaritiesbetween these molecules based on one of these factors, the use of allthree data points can be utilized to distinguish and differentiatebiochemicals. More than 3300 commercially available purified standardcompounds were acquired and registered for analysis on all platforms todetermine their analytical characteristics. Additional mass spectralentries were created for structurally unnamed biochemicals, which wereidentified by virtue of their recurrent nature (both chromatographic andmass spectral). Entries were further processed by manual curation toensure accurate and consistent identification of true chemical entities,and to remove those representing system artifacts, mis-assignments, andbackground noise.

Metabolite Quantification and Normalization

UPLC MS/MS peaks were quantified by area under the ROC curve (AUC). Datawere normalized to correct for variations resulting from instrumentinter-day tuning differences using median-centered method. In thismethod, each compound was corrected in run-day blocks by registering themedian as 1.00 and normalizing each data point proportionately,hereafter referred to as block correction, and further normalized toaccount for differences in metabolite levels due to differences in thequantities of material in each sample.

Differentially Abundant Metabolite Identification

Metabolites differentially abundant across all samples from the allergicand healthy twin groups (n=36) were identified using Welch's two-samplet-test. A total of 1,308 metabolites were quantified. After removingmetabolites without pathway annotation, 992 metabolites were kept forstatistical comparisons. Among those, in comparing allergic with healthygroups, 97 metabolites reached a significance level of P<0.10 and werekept for further analysis. After BH-method correction for multipletesting, none of the metabolites passed FDR 0.10, potentially due tosmall sample size. Additionally, the abundance of metabolites wascompared between the two groups within discordant twins only (n=26, from13 twin pairs) using paired t-test. The sample (S5077) and thecorresponding twin pair (#13) excluded from microbial 16S data analysiswas kept in the metabolic profiling analysis.

Correlation of Bacterial Taxa and Metabolite Abundance

Pairwise Spearman's rank correlation was computed among the 64 OTUs and97 metabolites differentially abundant between allergic and healthytwins (FIG. 17 ). OTUs were further prioritized using the followingapproach. First, OTUs were filtered for those that showed a correlationat P<0.05 with at least 5 differentially abundant metabolites from thedesignated group. For potentially healthy-abundant OTUs (more abundantin the healthy group), they were correlated with metabolites from group“Up in healthy” or “Down in healthy”; the potentially allergic-abundantOTUs (more abundant in allergic group) were correlated with metabolitesfrom group “Up in allergic” or “Down in allergic.” Second, OTUs thatpassed step 1 were filtered further for those that showed a relativelyconsistent trend of positive correlation (Spearman's p>0.20) across atleast 30% of the metabolites from the designated group. Forhealthy-abundant OTUs, they were correlated with metabolites from group“Up in healthy” and “Down in allergic” joined; the allergic-abundantOTUs were correlated with metabolites from group “Up in allergic” and“Down in healthy” together. Third, OTUs that passed steps 1 and 2 werefurther filtered to select those at P<0.05 from the DS-FDR methodcomparing allergic to healthy groups across all samples. Of the 64 OTUs,22 passed these correlation filters and are shown in FIG. 18 . After aBLAST search of assembled 16S sequences against the NCBI database (16Sribosomal RNA, Bacteria and Archaea) (accessed 09/12/2020) using blastn(20), 3 of 22 OTUs were matched to bacterial species at 99% or highersequence identity: OTU556835, matched to Phascolarctobacterium faecium(accession ID NR_026111.1, 99.21% identity); OTU188079 and OTU823634,both matched Ruminococcus bromii (accession ID NR_025930.1; 99.21%identity).

Statistics

The Discrete False-Discovery Rate (DS-FDR) method (29) was used toidentify differentially abundant OTUs by comparing allergic to healthytwins. Welch's two-sample t-test was used to identify differentiallyabundant metabolites comparing allergic to healthy twins. Unless statedotherwise, for comparing groups using all samples, Wilcoxon rank-sumtest was used; if only within discordant twins, Wilcoxon signed-ranktest was used. For metabolites, paired t-test was used to compare itsabundance between the two groups within discordant twins after log 10transformation. metabolic sub-pathway enrichment was analyzed using theHypergeometric test, requiring at least 2 metabolites annotated witheach sub-pathway. Following Wilcoxon rank-sum test or Wilcoxonsigned-rank test, and Hypergeometric test, we used the BH-FDR method(31) for multiple testing correction. For pairwise comparisons ofmetabolite Spearman's correlation coefficients between OTU clusters,Tukey's honestly significant difference (HSD) test was used. Otherstatistical tests were used as indicated in the text and in the BriefDescription of the Drawings. For comparison of healthy and allergicgroups across all samples in 16S analysis, qPCR validation, andSpearman's correlation between OTUs and metabolites, a P-value less than0.05 was considered significant. For comparison of healthy and allergicgroups across all samples in metabolite analysis, comparison of healthyand allergic groups within discordant twin pairs in 16S or metaboliteanalysis, and SCFA analysis, a P-value less than 0.10 was consideredsignificant. For metabolite sub-pathway enrichment analysis, anFDR-adjusted P-value less than 0.10 was considered significant. Alltests were two-sided.

Results

Bacteria produce many metabolites that modulate the immune system andprofoundly influence human health (32). Limited data exist on unbiasedsystematic profiling of fecal metabolites in patients with and withoutfood allergy. LC-MS/MS was performed to measure the abundance ofcompounds in the same set of fecal samples from the twin cohort. 97metabolites were identified as differentially abundant between thehealthy and allergic twins, with 33 more abundant in healthy twins, and64 more abundant in allergic twins (FIGS. 13A and 131B; Table 4). Whenstatistical comparisons were restricted to monozygotic twins only (14pairs, 28 samples), the test statistics for the 33 healthy-abundantmetabolites and the 64 allergic-abundant metabolites correlated withthat of all samples (18 pairs, 36 samples) (FIG. 12 , Table 4). Amongthese 97 metabolites, 32 (16 higher in healthy, 16 higher in allergic)also reached a significance level of 0.10 within discordant twin pairsonly (FIGS. 16A-16H).

TABLE 4 List of 97 metabolites with known pathway annotation identifiedherein as differentially abundant between healthy and allergic twins CASMass Biochemical Name Pubchem ID 60-27-5 114.0662 creatinine 588 58-85-5245.0955 biotin 171548 110-60-1 89.1073 putrescine 1045 621-37-4151.0401 3-hydroxyphenylacetate 12122 107-35-7 126.022 taurine 112358052-48-5 169.0972 pyridoxamine 1052 7432-23-7 311.1238gamma-glutamyltyrosine 94340 107-43-7 118.0863 betaine 247 462-88-4133.0608 3-ureidopropionate 111 64-04-0 122.0964 phenethylamine 10011476-39-7; 191.1026 diaminopimelate 439283 2577-62-0 207300-70-7193.0354 glucuronate 444791 15763-06-1 282.1197 1-methyladenosine 27476601-75-2 131.035 ethylmalonate 11756 102-32-9 123.04523,4-dihydroxyphenylacetate 547 145-42-6 514.2844 taurocholate 667513545-04-5 145.0506 2,3-dimethylsuccinate 11848 57-00-1 132.0768creatine 586 583-50-6 119.035 erythrose 94176 279.6142 taurocholenatesulfate* NA 83-34-1 130.0662 skatol 6736 119-13-1 402.3492delta-tocopherol 92094 500-98-1 192.0666 phenylacetylglycine 68144155-84-0 217.1295 N-acetylarginine 67427 2430-94-6 197.15475-dodecenoate (12:1n7) 5312378 474-62-4 383.3672 campesterol 173183124151-74-2 277.2173 1-linolenoylglycerol (18:3) 53480978 570.34132-palmitoyl-GPC* (16:0)* 15061532 4918-96-1 188.9863 catechol sulfate3083879 64936-83-0 280.6221 taurolithocholate 3-sulfate 44007140632-52-8 305.018 uridine-2′,3′-cyclic monophosphate 439715 15718-49-7344.0402 guanosine-2′,3′-cyclic monophosphate 92823 371.18985alpha-androstan-3beta, N/A 17alpha-diol monosulfate (1) 15718-51-1304.034 cytidine 2′,3′-cyclic monophosphate 417654 29388-59-8 361.1657secoisolariciresinol 65373 95-55-6 110.06 2-aminophenol 5801 20316-62-5593.1301 tribuloside 5320686 263399-34-4 313.2384 9,10-DiHOME 99666401119-48-8 118.0863 N-methyl-GABA 70703 52514-04-2 388.2527N-oleoyltaurine 6437033 3947-38-4 589.3021 D-urobilin 6276321148244-82-0 685.2713 secoisolariciresinol diglucoside 9917980 491-71-4299.0561 chrysoeriol 5280666 11034-77-8 343.2279 (15:2)-anacardic acid11824131 501-94-0 137.0608 tyrosol 10393 15450-76-7 162.0552,8-quinolinediol 97250 107-68-6 138.023 N-methyltaurine 7882 501-75-7126.1026 1-methylhistamine 3614 1164-98-3 245.067121-hydroxypregnenolone disulfate 134595 203.002 4-methylcatechol sulfateN/A 166.018 N-acetyltaurine 159864 20245-33-4 283.1037 N1-methylinosine65095 232.0285 dopamine 3-O-sulfate 122136 74509-14-1 253.1194 pyrraline122228 40712-60-5 230.0129 2-acetamidophenol sulfate 181671 239.99722,8-quinolinediol sulfate N/A 204.9812 1,2,3-benzenetriol sulfate (1)N/A 204.9812 1,2,3-benzenetriol sulfate (2) N/A 7235-40-7 552.4312beta-carotene 5280489 56421-10-4 788.6164 l-stearoyl-2-oleoyl-GPC(18:0/18:1) N/A 416.3643 gamma-tocopherol/beta-tocopherol N/A 610.5405palmitoyl-linoleoyl-glycerol 9543695 (16:0/18:2) [1]* 610.5405palmitoyl-linoleoyl-glycerol N/A (16:0/18:2) [2]* 398.3265palmitoleoylcamitine (C 16:l)* 71464547 258.9918 caffeic acid sulfateN/A 923-42-2 189.0405 3-carboxyadipate 222467 56392-16-6 284.1856alpha-hydroxymetoprolol 114962 507.2728 1-linoleoyl-GPG (18:2)* NA2475-56-1 211.0612 vanillactate 160637 55304-02-4 795.4536 soyasaponinIII N/A 638.5718 stearoyl-linoleoyl-glycerol N/A (18:0/18:2) [2]*630.5092 linolenoyl-linolenoyl-glycerol N/A (18:3/18:3) [1]* 630.5092linolenoyl-linolenoyl-glycerol N/A (18:3/18:3) [2]* 632.5249linoleoyl-linolenoyl-glycerol N/A (18:2/18:3) [1]* 632.5249linoleoyl-linolenoyl-glycerol N/A (18:2/18:3) [2]* 608.5249palmitoyl-linolenoyl-glycerol N/A (16:0/18:3) [2]* 634.5405linoleoyl-linoleoyl-glycerol N/A (18:2/18:2) [2]* 247.0925succinylglutamine N/A 356.3523 arachidoyl ethanolamide (20:0)* 3787294384.3836 behenoyl ethanolamide (22:0)* 3023585 14868-24-7 133.05062,3-dihydroxy-2-methylbutyrate 301941 78919-26-3 391.2854isoursodeoxycholate 127601 568.4279 carotene diol (3) N/A 7561-64-0249.186 hexadecatrienoate (16:3n3) 5312428 305.1456gamma-glutamylcitrulline* N/A 352.086 sulfate of piperine metabolite N/AC16H19NO3 (2)* 352.086 sulfate of piperine metabolite N/A C16H19NO3 (3)*261.0074 dihydrocaffeate sulfate (2) 49844181 229.1547N,N,N-trimethyl-alanylproline N/A betaine (TMAP) 1518-62-3 119.0352,4-dihydroxybutyrate 192742 411.3622 stigmastadienone 6442194 222.0806hexanoyltaurine 2245940 165.0405 pentose acid* N/A 40165-89-7 285.24353-hydroxymargarate 93220 17860-87-6 189.0405 2-O-methylascorbic acid99779 377.07 enterolactone sulfate N/A 60-27-5 218.99692-methoxyhydroquinone sulfate (1) N/A

After annotating the 97 metabolites into super-pathways andsub-pathways, healthy twins showed distinct enrichment at the pathwaylevel compared with allergic twins (FIG. 14A). Specifically, as shown inFIG. 14A, among other pathways, the diacylglycerol (DAG) sub-pathway wassignificantly enriched in the 33 metabolites more abundant in healthytwins (FDR-adjusted P<0.00001), and food component/plant sub-pathway wassignificantly enriched in the 64 metabolites more abundant in allergictwins (FDR-adjusted P=0.0074). One of the DAG metabolites,Linoleoyl-linolenoyl-glycerol (18:2/18:3) [1]*(Comp ID: 54963), wassignificantly higher (P=0.0036) in healthy twins compared to allergictwins in discordant pairs (FIG. 15A) and was significant in the twincohort overall (FIG. 14B, P=0.019). On the other hand,secoisolariciresinol (Comp ID: 38105) (SECO) from the foodcomponent/plant pathway was higher in allergic twins compared withhealthy twins (P=0.0067) (FIG. 14C) with the same trend observed indiscordant twin pairs (P=0.094) (FIG. 15B).

In an attempt to interpret the source of these metabolites, the data wascompared with an internal microbial metabolite database under activedevelopment from Metabolon Inc. (accessed Oct. 24, 2019). Among the 992metabolites with annotated pathways that were examined, 129 overlappedwith the Metabolon database. Of the 129 overlapping metabolites, 66 weremarked with discovery sites such as colon, feces, urine, plasma,tissues, or multiple sites; 38 (58%) of the 66 metabolites were fromcolon or feces. Also, of the 129 metabolites, 13 were among the 97compounds significantly differentially abundant between healthy andallergic twins, including 1-methylhistamine, 3-hydroxyphenylacetate,3,4-dihydroxyphenylacetate, betaine, skatol, ethylmalonate, creatine,creatinine, putrescine, phenylacetylglycine, taurolithocholate3-sulfate, biotin, and D-urobilin.

Additionally, eight short-chain fatty acids (SCFAs) were profiled usingGC-MS technology: 2-methylbutyric acid, acetic acid, butyric acid,hexanoic acid, isobutyric acid, isovaleric acid, propionic acid, andvaleric acid. The abundance of these eight SCFAs was then comparedbetween allergic and healthy twins. Within the 13 discordant twin pairs,no SCFA reached P<0.10, potentially due to the single point-in-timeanalysis (data not shown).

The DAG sub-pathway was the most significantly different between healthyand allergic twins (FDR-adjusted P<0.00001) and was enriched in the 33metabolites more abundant in healthy twins. In addition to P. faecium(cluster 1), metabolites in the DAG sub-pathway were strongly correlatedwith other bacteria in OTU clusters 1 and 2, many of which areClostridia. The identification of readily measurable metabolites whichdistinguish healthy and allergic twins has important implications forthe development of microbiome-modulating therapeutics because of theirpotential as biomarkers, particularly in clinical trials.Laboratory-based assays measuring, in particular, DAG may have greatutility as biochemical indicators of therapeutic interventions thatshift the microbiota towards health.

The food component/plant pathway was most significantly enriched inallergic twins after FDR correction (FDR-adjusted P=0.0074),particularly the metabolite secoisolariciresinol (SECO). SECO iscommonly observed as an intermediate product in the bacteria-mediatedbreakdown of plant-derived lignans into enterolignans, such asenterodiol and enterolactone, which have numerous benefits for humanhealth (33-35). Several bacterial species and genes are involved in themulti-step process of lignan metabolism (34, 36, 37). Of particularinterest, the gene glm codes for an enzyme which methylates SECO intodmSECO, allowing further biotransformation into enterodiol by otherbacteria (37) Phylogenetic analysis suggests that glm is expressed by awide variety of bacteria, the majority of which are Lachnospiraceae(37). The abundance of SECO in feces was negatively correlated with theabundance of several OTUs in healthy individuals (many of which wereLachnospiraceae or Ruminococcaceae). The high abundance of SECO in thefeces of allergic twins supports the taxonomic analysis, as the buildupof this intermediate product may be a direct effect of the lowerabundance of Clostridia in these individuals.

Example 3—Identifying Bacterial Species with a Protective Role AgainstFood Allergy

The two datasets described in Example 1 (OTUs) and Example 2(metabolites) were correlated to identify any bacterial species ormetabolites that may be mechanistically related to health in the cohort.Overall, the OTUs differentially abundant between healthy and allergictwin groups were correlated with different sets of metabolites andpathways. The abundance of 64 differentially abundant OTUs wascorrelated with the 97 metabolites (FIG. 17 ) and 21 healthy-abundantOTUs and one allergic-abundant OTU were identified with consistentcorrelation across metabolites at per sample level (FIG. 18 ; Table 5).

TABLE 5 List of 22 OTUs correlated with metabolites OTU Family 186478Ruminococcaceae 188079 Ruminococcaceae 196139 Ruminococcaceae 198184Ruminococcaceae 509709 Lachnospiraceae 556835 Veillonellaceae 574038Lachnospiraceae 658370 Lachnospiraceae 823634 RuminococcaceaeNew.CleanUp.ReferenceOTU112566 BacteroidaceaeNew.CleanUp.ReferenceOTU1320 RuminococcaceaeNew.CleanUp.ReferenceOTU135990 LachnospiraceaeNew.CleanUp.ReferenceOTU141755 LachnospiraceaeNew.CleanUp.ReferenceOTU153408 BacteroidaceaeNew.CleanUp.ReferenceOTU153961 RuminococcaceaeNew.CleanUp.ReferenceOTU164203 RuminococcaceaeNew.CleanUp.ReferenceOTU28435 LachnospiraceaeNew.CleanUp.ReferenceOTU57003 LachnospiraceaeNew.CleanUp.ReferenceOTU58395 LachnospiraceaeNew.CleanUp.ReferenceOTU86790 LachnospiraceaeNew.CleanUp.ReferenceOTU92834 Ruminococcaceae New.ReferenceOTU129Bacteroidaceae

The metabolites were divided into 5 categories based on their abundancecorrelation consistency among OTU clusters 1 to 3 (consisting of 21healthy-abundant OTUs; cluster 4 only contains 1 OTU from anallergic-abundant taxon and was not used for metabolite groupannotation). The 5 metabolite groups are (FIG. 18 ; FIG. 19 ): (group 1)positively correlated with the 3 OTU clusters, stronger in clusters 1and 2 relative to cluster 3 (n=9); (group 2) positively correlated withthe 3 OTU clusters, stronger in cluster 3 relative to clusters 1 and 2(n=8); (group 3) correlated with OTU clusters with mixed patterns(n=16); (group 4) negatively correlated with the 3 OTU clusters,stronger in cluster 1 relative to clusters 2 and 3 (n=46); and (group 5)negatively correlated with the 3 OTU clusters with similar distribution(n=22). These 5 metabolite groups showed distinctly differentdistributions of metabolite super-pathways and sub-pathways (FIG. 20A).In particular, group 1 was dominated by metabolites from lipidsuper-pathway including diacylglycerol and monoacylglycerol (FIG. 20A),whereas whereas amino acid metabolism, including tyrosine,phenylalanine, arginine, proline, methionine, cysteine,S-adenosylmethionine (SAM), and taurine was enriched in group 2 (FIG.20A).

To annotate the 22 OTUs at the species-level resolution, the assembled16S sequence of each OTU was searched against NCBI's Bacteria/Archaea16S reference database using BLAST (34). At sequence identity of 99% orhigher, OTU556835 was matched to Phascolarctobacterium faecium(accession ID NR_026111.1) and both OTU188079 and OTU823634 were matchedto Ruminococcus bromii (accession ID NR_025930.1). The other OTUs(abundant in either healthy or allergic twins) did not have matchesmeeting the identity threshold. Quantitative PCR (qPCR) validated thesignificantly higher abundance of P. faecium in healthy twins comparedwith allergic twins (P=0.016) (FIGS. 20B and 20C; FIG. 21B). P. faeciumis an obligate anaerobic non-spore-forming bacterium that consumessuccinate and produces SCFAs including acetate and propionate (17, 18).P. faecium was grouped in cluster 1 and was most highly correlated witha number of DAG metabolites. P. faecium was also strongly positivelycorrelated with tocopherol and negatively correlated with a variety ofmetabolites, including those from the secondary bile acid metabolismpathway. R. bromii was also qPCR-validated to be enriched in the healthycompared with the allergic twin group (P=0.022) (FIGS. 20D and 20E; FIG.22B). R. bromii is a strictly anaerobic, spore-forming Clostridiaimportant for the degradation of dietary resistant starch (19). It wasassociated with group 2 metabolites involved in fatty acid, amino acid,and sterol metabolism.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of certain embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A method for preventing or reducing an immune response to an allergenin a subject, the method comprising administering to the subject acomposition comprising a therapeutically effective amount of (a)Phascolarctobacterium faecium or (b) Ruminococcus bromii.
 2. The methodof claim 1, wherein the subject is at risk for an anaphylactic responseto the allergen.
 3. The method of claim 1 or 2, wherein the compositioncomprises Phascolarctobacterium faecium.
 4. The method of claim 3,wherein the composition comprises between 1×10³ and 1×10¹⁵ colonyforming units (CFU) of Phascolarctobacterium faecium.
 5. The method ofany of claims 1-4, wherein the composition comprises Ruminococcusbromii.
 6. The method of claim 5, wherein the composition comprisesbetween 1×10³ and 1×10¹⁵ CFU of Ruminococcus bromii.
 7. The method ofany of claims 1-6, wherein the Phascolarctobacterium faecium orRuminococcus bromii make up at least 75% of all bacteria in thecomposition.
 8. The method of claim 7, wherein the Phascolarctobacteriumfaecium or Ruminococcus bromii make up at least 95% of all bacteria inthe composition.
 9. The method of claim 8, wherein thePhascolarctobacterium faecium or Ruminococcus bromii make up about 100%of all bacteria in the composition.
 10. The method of any of claims 1-9,wherein the composition comprises Phascolarctobacterium faecium andRuminococcus bromii.
 11. The method of claim 10, wherein the compositioncomprises (i) between 1×10³ and 1×10¹⁵ CFU of Phascolarctobacteriumfaecium and (ii) between 1×10³ and 1×10¹⁵ CFU of Ruminococcus bromii.12. The method of any of claims 1-11, wherein the composition does notcomprise more than a contaminating amount of any other bacteria.
 13. Themethod of any of claims 1-11, wherein the composition does not comprisea detectable amount of any other bacteria.
 14. The method of any ofclaims 1-13. wherein the composition further comprises one or moremicroparticles.
 15. The method of claim 14, wherein thePhascolarctobacterium faecium or Ruminococcus bromii are encapsulatedwithin the one or more microparticles.
 16. The method of claim 14,wherein the Phascolarctobacterium faecium or Ruminococcus bromii are notencapsulated within the one or more microparticles.
 17. The method ofany of claims 1-16, wherein the allergen is a food allergen.
 18. Themethod of any of claims 1-17, wherein the subject has a food allergy.19. The method of any of claims 1-17, wherein the subject is at risk ofa food allergy.
 20. The method of any of claims 1-19, wherein thesubject was determined to have symptoms of a food allergy.
 21. Themethod of any of claims 1-19, wherein the subject was diagnosed with afood allergy.
 22. The method of any of claims 1-19, wherein the subjectwas not diagnosed with a food allergy.
 23. The method of any of claims19-22, wherein the food allergy is an allergy to one or more of peanuts,tree nuts, shellfish, soy, egg, fish, mustard, oats, olives, corn, rice,pineapple, wheat, gluten, milk, sesame, garbanzo beans, bananas, kiwi,avocado, mangos, melons, carrots, cucumber, apples, squash, and crab.24. The method of any of claims 19-23, wherein the subject has beenpreviously treated for a food allergy.
 25. The method of claim 24,wherein the subject was determined to be resistant to the previoustreatment.
 26. The method of any of claims 19-23, wherein the subjecthas not been previously treated for a food allergy.
 27. The method ofany of claims 1-26, wherein the composition comprises a bacterialproduct comprising the Phascolarctobacterium faecium or Ruminococcusbromii.
 28. The method of claim 27, wherein the bacterial productcomprises Phascolarctobacterium faecium and Ruminococcus bromii.
 29. Themethod of claim 27 or 28, wherein the bacterial product is a livebacterial product.
 30. The method of any of claims 27-29, wherein thebacterial product is a lyophilized or freeze-dried bacterial product.31. The method of any of claims 27-30, wherein the bacterial product isa bacterial product isolated from a human subject.
 32. The method of anyof claims 27-30, wherein the bacterial product is a bacterial productisolated from a non-human subject.
 33. The method of any of claims 1-32,wherein the composition is administered orally.
 34. The method of any ofclaims 1-33, wherein the method comprises preventing an anaphylacticresponse in the subject.
 35. The method of any of claims 1-34, furthercomprising providing to the subject a prebiotic.
 36. The method of claim35, wherein the prebiotic comprises one or more ofgalactooligosacchararide, lactulose, lactitol, erythritol, isomalt,polyglycitol, and succinate.
 37. The method of claim 35, wherein theprebiotic comprises one or more of galactooligosacchararide, lactulose,succinate, and lactitol.
 38. The method of claim 35, wherein theprebiotic is a resistant starch.
 39. The method of claim 35, wherein theprebiotic is a potato starch.
 40. The method of any of claims 35-39,wherein the prebiotic comprises one or more of a digestible and anon-digestible oligosaccharide.
 41. The method of any of claims 35-40,wherein at least 10 grams of prebiotic is administered to the subject.42. The method of any of claims 35-41, wherein the ratio of the colonyforming units of the Phascolarctobacterium faecium or Ruminococcusbromii to grams of prebiotic is 1000:1-10000:1.
 43. The method of any ofclaims 1-42, wherein the subject was determined to have a decreasedoperational taxonomic units (OTU) abundance score relative to a controlor reference sample, wherein the OTU abundance score was calculatedusing at least 20 of the OTUs of Table
 1. 44. The method of claim 43,wherein the OTU abundance score was calculated using at least 30 of theOTUs of Table
 1. 45. The method of claim 44, wherein the OTU abundancescore was calculated using at least 40 of the OTUs of Table
 1. 46. Themethod of claim 45, wherein the OTU abundance score was calculated usingat least 50 of the OTUs of Table
 1. 47. The method of claim 46, whereinthe OTU abundance score was calculated using at least 60 of the OTUs ofTable
 1. 48. The method of claim 47, wherein the OTU abundance score wascalculated using all of the OTUs of Table
 1. 49. The method of any ofclaims 43-48, wherein the control or reference sample is a sample from ahealthy individual.
 50. The method of any of claims 1-49, wherein thesubject was determined to have a decreased abundance of metabolites froma metabolic pathway, wherein the metabolic pathway is the diacylglycerolpathway, the sterol pathway, the tocopherol metabolism pathway, thegamma-glutamyl amino acid pathway, or the endocannabinoid pathway. 51.The method of claim 50, wherein the metabolic pathway is thediacylglycerol pathway.
 52. The method of any of claims 1-51, whereinthe subject was determined to have a decreased abundance oflinoleoyl-linoleoyl-glycerol (18:2/18:3).
 53. The method of any ofclaims 50-52, wherein the decreased abundance was determined from afecal sample from the subject.
 54. The method of any of claims 1-53,wherein the subject was determined to have an increased abundance ofmetabolites from a metabolic pathway, wherein the metabolic pathway isthe creatine metabolism pathway, the dihydroxy fatty acid pathway, thetyrosine metabolism pathway, or the food component/plant metabolismpathway.
 55. The method of any of claims 1-54, wherein the subject wasdetermined to have an increased abundance of secoisolariciresinol. 56.The method of claim 54 or 55, wherein the increased abundance wasdetermined from a fecal sample from the subject.
 57. The method of anyof claims 1-56, further comprising administering the allergen to thesubject.
 58. The method of claim 57, wherein the composition isadministered prior to the allergen.
 59. The method of claim 58, whereinthe composition is administered at most 24 hours prior to the allergen.60. The method of claim 59, wherein the composition is administered atmost 12 hours prior to the allergen.
 61. The method of claim 60, whereinthe composition is administered at most 6 hours prior to the allergen.62. The method of any of claims 1-56, wherein the composition isadministered after the allergen.
 63. The method of any of claim 62,wherein the composition is administered at most 24 hours after theallergen.
 64. The method of any of claim 63, wherein the composition isadministered at most 12 hours after the allergen.
 65. The method of anyof claim 64, wherein the composition is administered at most 6 hoursafter the allergen.
 66. The method of any of claims 1-65, furthercomprising providing an immunotherapy to the subject.
 67. The method ofclaim 66, wherein the immunotherapy is an oral immunotherapy.
 68. Themethod of claim 66, wherein the immunotherapy is an epicutaneousimmunotherapy.
 69. The method of any of claims 66-68, wherein theimmunotherapy is provided prior to administering the composition to thesubject.
 70. The method of any of claims 66-68, wherein theimmunotherapy is provided after administering the composition to thesubject.
 71. A method for diagnosing a subject with a food allergy, themethod comprising determining the subject to have a decreased OTUabundance score relative to a control or reference sample, wherein theOTU abundance score is calculated using at least 20 of the OTUs of Table1, thereby diagnosing the subject with the food allergy.
 72. The methodof claim 71, wherein the OTU abundance score is calculated using atleast 30 of the OTUs of Table
 1. 73. The method of claim 72, wherein theOTU abundance score is calculated using at least 40 of the OTUs ofTable
 1. 74. The method of claim 73, wherein the OTU abundance score iacalculated using at least 50 of the OTUs of Table
 1. 75. The method ofclaim 74, wherein the OTU abundance score ia calculated using at least60 of the OTUs of Table
 1. 76. The method of claim 75, wherein the OTUabundance score is calculated using all of the OTUs of Table
 1. 77. Themethod of any of claims 71-76, wherein the control or reference sampleis a sample from a healthy individual.
 78. The method of any of claims71-77, further comprising determining the subject to have a decreasedabundance of metabolites from a metabolic pathway, wherein the metabolicpathway is the diacylglycerol pathway, the sterol pathway, thetocopherol metabolism pathway, the gamma-glutamyl amino acid pathway, orthe endocannabinoid pathway.
 79. The method of claim 78, wherein themetabolic pathway is the diacylglycerol pathway.
 80. The method of anyof claims 71-79, further comprising determining the subject to have adecreased abundance of linoleoyl-linoleoyl-glycerol (18:2/18:3).
 81. Themethod of any of claims 78-80, wherein the decreased abundance isdetermined from a fecal sample from the subject.
 82. The method of anyof claims 71-81, further comprising determining the subject to have anincreased abundance of metabolites from a metabolic pathway, wherein themetabolic pathway is the creatine metabolism pathway, the dihydroxyfatty acid pathway, the tyrosine metabolism pathway, or the foodcomponent/plant metabolism pathway.
 83. The method of any of claims71-82, further comprising determining the subject to have an increasedabundance of secoisolariciresinol.
 84. The method of claim 82 or 83,wherein the increased abundance is determined from a fecal sample fromthe subject.
 85. A method for treating a food allergy in a subject, themethod comprising providing to the subject a food allergy therapy,wherein the food allergy therapy is: an immunotherapy, a steroid, anantihistamine, a hormone, a microbiome-modulating therapy, or acombination thereof, wherein the subject was determined to have adecreased OTU abundance score relative to a control or reference sample,and wherein the OTU abundance score was calculated using at least 20 ofthe OTUs of Table
 1. 86. The method of claim 85, wherein the OTUabundance score was calculated using at least 30 of the OTUs of Table 1.87. The method of claim 86, wherein the OTU abundance score wascalculated using at least 40 of the OTUs of Table
 1. 88. The method ofclaim 87, wherein the OTU abundance score was calculated using at least50 of the OTUs of Table
 1. 89. The method of claim 88, wherein the OTUabundance score was calculated using at least 60 of the OTUs of Table 1.90. The method of claim 87, wherein the OTU abundance score wascalculated using all of the OTUs of Table
 1. 91. The method of any ofclaims 85-90, wherein the control or reference sample is a sample from ahealthy individual.
 92. The method of any of claims 85-91, wherein thesubject has a food allergy.
 93. The method of any of claims 85-91,wherein the subject is at risk of a food allergy.
 94. The method ofclaim 93, wherein the food allergy is an allergy to one or more ofpeanuts, tree nuts, shellfish, soy, egg, fish, mustard, oats, olives,corn, rice, pineapple, wheat, gluten, milk, sesame, garbanzo beans,bananas, kiwi, avocado, mangos, melons, carrots, cucumber, apples,squash, and crab.
 95. The method of any of claims 85-94, wherein thefood allergy therapy is a microbiome-modulating therapy.
 96. The methodof claim 95, wherein the microbiome-modulating therapy comprisesproviding to the subject a composition comprising a therapeuticallyeffective amount of (a) Phascolarctobacterium faecium or (b)Ruminococcus bromii.
 97. The method of claim 96, wherein the compositioncomprises Phascolarctobacterium faecium.
 98. The method of claim 97,wherein the composition comprises between 1×10³ and 1×10¹⁵ CFU ofPhascolarctobacterium faecium.
 99. The method of claim 96, wherein thecomposition comprises Ruminococcus bromii.
 100. The method of claim 99,wherein the composition comprises between 1×10³ and 1×10¹⁵ CFU ofRuminococcus bromii.
 101. The method of any of claims 96-100, whereinthe Phascolarctobacterium faecium or Ruminococcus bromii make up atleast 75% of all bacteria in the composition.
 102. The method of claim101, wherein the Phascolarctobacterium faecium or Ruminococcus bromiimake up at least 95% of all bacteria in the composition.
 103. The methodof claim 102, wherein the Phascolarctobacterium faecium or Ruminococcusbromii make up about 100% of all bacteria in the composition.
 104. Themethod of any of claims 96-103, wherein the composition comprisesPhascolarctobacterium faecium and Ruminococcus bromii.
 105. The methodof claim 104, wherein the composition comprises (i) between 1×10³ and1×10¹⁵ CFU of Phascolarctobacterium faecium and (ii) between 1×10³ and1×10¹⁵ CFU of Ruminococcus bromii.
 106. The method of any of claims96-105, wherein the composition does not comprise more than acontaminating amount of any other bacteria.
 107. The method of any ofclaims 96-105, wherein the composition does not comprise a detectableamount of any other bacteria.
 108. The method of any of claims 96-107,wherein the composition further comprises one or more microparticles.109. The method of claim 108, wherein the Phascolarctobacterium faeciumor Ruminococcus bromii are encapsulated within the one or moremicroparticles.
 110. The method of claim 108, wherein thePhascolarctobacterium faecium or Ruminococcus bromii are notencapsulated within the one or more microparticles.
 111. The method ofany of claims 96-110, wherein the composition comprises a bacterialproduct comprising the Phascolarctobacterium faecium or Ruminococcusbromii.
 112. The method of claim 111, wherein the bacterial productcomprises Phascolarctobacterium faecium and Ruminococcus bromii. 113.The method of claim 111 or 112, wherein the bacterial product is a livebacterial product.
 114. The method of any of claims 111-113, wherein thebacterial product is a lyophilized or freeze-dried bacterial product.115. The method of any of claims 111-114, wherein the bacterial productis a bacterial product isolated from a human subject.
 116. The method ofany of claims 111-115, wherein the bacterial product is a bacterialproduct isolated from a non-human subject.
 117. The method of any ofclaims 96-116, wherein the composition is administered orally.
 118. Themethod of any of claims 96-117, wherein the composition furthercomprises a prebiotic.
 119. The method of claim 118, wherein theprebiotic comprises one or more of galactooligosacchararide, lactulose,lactitol, erythritol, isomalt, polyglycitol, and succinate.
 120. Themethod of claim 118, wherein the prebiotic comprises one or more ofgalactooligosacchararide, lactulose, succinate, and lactitol.
 121. Themethod of claim 118, wherein the prebiotic is a resistant starch. 122.The method of claim 118, wherein the prebiotic is a potato starch. 123.The method of any of claims 118-122, wherein the prebiotic comprises oneor more of a digestible and a non-digestible oligosaccharide.
 124. Themethod of any of claims 118-123, wherein at least 10 grams of prebioticis administered to the subject.
 125. The method of any of claims118-124, wherein the ratio of the colony forming units of thePhascolarctobacterium faecium or Ruminococcus bromii to grams ofprebiotic in the composition is 1000:1-10000:1.
 126. The method of anyof claims 96-125, wherein the composition is administered with a food ofthe food allergy.
 127. The method of claim 126, wherein the compositionis administered prior to the food.
 128. The method of claim 127, whereinthe composition is administered at most 24 hours prior to the food. 129.The method of claim 128, wherein the composition is administered at most12 hours prior to the food.
 130. The method of claim 129, wherein thecomposition is administered at most 6 hours prior to the food.
 131. Themethod of claim 126, wherein the composition is administered after thefood.
 132. The method of claim 131, wherein the composition isadministered at most 24 hours after the food.
 133. The method of claim132, wherein the composition is administered at most 12 hours after thefood.
 134. The method of claim 133, wherein the composition isadministered at most 6 hours after the food.
 135. The method of any ofclaims 85-134, further comprising providing an immunotherapy to thesubject.
 136. The method of claim 135, wherein the immunotherapy is anoral immunotherapy.
 137. The method of claim 135, wherein theimmunotherapy is an epicutaneous immunotherapy.
 138. The method of anyof claims 135-137, wherein the immunotherapy is provided prior toadministering the composition to the subject.
 139. The method of any ofclaims 135-137, wherein the immunotherapy is provided afteradministering the composition to the subject.
 140. The method of any ofclaims 85-139, wherein the subject was further determined to have adecreased abundance of metabolites from a metabolic pathway, wherein themetabolic pathway is the diacylglycerol pathway, the sterol pathway, thetocopherol metabolism pathway, the gamma-glutamyl amino acid pathway, orthe endocannabinoid pathway.
 141. The method of claim 140, wherein themetabolic pathway is the diacylglycerol pathway.
 142. The method of anyof claims 85-141, wherein the subject was further determined to have adecreased abundance of linoleoyl-linoleoyl-glycerol (18:2/18:3) relativeto a control to reference sample.
 143. The method of any of claims140-142, wherein the decreased abundance was determined from a fecalsample from the subject.
 144. The method of any of claims 85-143,wherein the subject was further determined to have an increasedabundance of metabolites from a metabolic pathway, wherein the metabolicpathway is the creatine metabolism pathway, the dihydroxy fatty acidpathway, the tyrosine metabolism pathway, or the food component/plantmetabolism pathway.
 145. The method of any of claims 85-144, wherein thesubject was further determined to have an increased abundance ofsecoisolariciresinol relative to a control to reference sample.
 146. Themethod of claim 144 or 145, wherein the increased abundance wasdetermined from a fecal sample from the subject.
 147. A method fortreating a food allergy in a subject, the method comprising providing tothe subject a food allergy therapy, wherein the food allergy therapy isan immunotherapy, a steroid, an antihistamine, a hormone, amicrobiome-modulating therapy, or a combination thereof, wherein thesubject was determined to have, relative to a control or referencesample: (a) a decreased abundance of metabolites from a metabolicpathway, wherein the metabolic pathway is the diacylglycerol pathway,the sterol pathway, the tocopherol metabolism pathway, thegamma-glutamyl amino acid pathway, or the endocannabinoid pathway, or(b) an increased abundance of metabolites from a metabolic pathway,wherein the metabolic pathway is the creatine metabolism pathway, thedihydroxy fatty acid pathway, the tyrosine metabolism pathway, or thefood component/plant metabolism pathway.
 148. The method of claim 147,wherein the subject was determined to have a decreased abundance ofmetabolites from the metabolic pathway, wherein the metabolic pathway isthe diacylglycerol pathway, the sterol pathway, the tocopherolmetabolism pathway, the gamma-glutamyl amino acid pathway, or theendocannabinoid pathway.
 149. The method of claim 148, wherein themetabolic pathway is the diacylglycerol pathway.
 150. The method of anyof claims 147-149, wherein the subject was determined to have adecreased abundance of linoleoyl-linoleoyl-glycerol (18:2/18:3). 151.The method of any of claims 147-150, wherein the subject was determinedto have an increased abundance of metabolites from the metabolicpathway, wherein the metabolic pathway is the creatine metabolismpathway, the dihydroxy fatty acid pathway, the tyrosine metabolismpathway, or the food component/plant metabolism pathway.
 152. The methodof claim 151, wherein the subject was determined to have an increasedabundance of secoisolariciresinol relative to a control to referencesample.
 153. The method of any of claims 147-152, wherein the increasedabundance or the decreased abundance was determined from a fecal samplefrom the subject.
 154. The method of any of claims 147-153, wherein thecontrol or reference sample is a sample from a healthy individual. 155.The method of any of claims 147-154, wherein the subject has or is atrisk of a food allergy.
 156. The method of claim 155, wherein the foodallergy is an allergy to one or more of peanuts, tree nuts, shellfish,soy, egg, fish, mustard, oats, olives, corn, rice, pineapple, wheat,gluten, milk, sesame, garbanzo beans, bananas, kiwi, avocado, mangos,melons, carrots, cucumber, apples, squash, and crab.
 157. The method ofany of claims 147-156, wherein the food allergy therapy is amicrobiome-modulating therapy.
 158. The method of claim 157, wherein themicrobiome-modulating therapy comprises providing to the subject acomposition comprising a therapeutically effective amount of (a)Phascolarctobacterium faecium or (b) Ruminococcus bromii.
 159. Themethod of claim 158, wherein the composition comprisesPhascolarctobacterium faecium.
 160. The method of claim 159, wherein thecomposition comprises between 1×10³ and 1×10¹⁵ CFU ofPhascolarctobacterium faecium.
 161. The method of claim 158, wherein thecomposition comprises Ruminococcus bromii.
 162. The method of claim 161,wherein the composition comprises between 1×10³ and 1×10¹⁵ CFU ofRuminococcus bromii.
 163. The method of any of claims 158-162, whereinthe Phascolarctobacterium faecium or Ruminococcus bromii make up atleast 75% of all bacteria in the composition.
 164. The method of claim163, wherein the Phascolarctobacterium faecium or Ruminococcus bromiimake up at least 95% of all bacteria in the composition.
 165. The methodof claim 164, wherein the Phascolarctobacterium faecium or Ruminococcusbromii make up about 100% of all bacteria in the composition.
 166. Themethod of any of claims 158-165, wherein the composition comprisesPhascolarctobacterium faecium and Ruminococcus bromii.
 167. The methodof claim 166, wherein the composition comprises (i) between 1×10³ and1×10¹⁵ CFU of Phascolarctobacterium faecium and (ii) between 1×10³ and1×10¹⁵ CFU of Ruminococcus bromii.
 168. The method of any of claims158-167, wherein the composition does not comprise more than acontaminating amount of any other bacteria.
 169. The method of any ofclaims 158-167, wherein the composition does not comprise a detectableamount of any other bacteria.
 170. The method of any of claims 158-169,wherein the composition further comprises one or more microparticles.171. The method of claim 170, wherein the Phascolarctobacterium faeciumor Ruminococcus bromii are encapsulated within the one or moremicroparticles.
 172. The method of claim 170, wherein thePhascolarctobacterium faecium or Ruminococcus bromii are notencapsulated within the one or more microparticles.
 173. The method ofany of claims 158-172, wherein the composition comprises a bacterialproduct comprising the Phascolarctobacterium faecium or Ruminococcusbromii.
 174. The method of claim 173, wherein the bacterial productcomprises Phascolarctobacterium faecium and Ruminococcus bromii. 175.The method of claim 173 or 174, wherein the bacterial product is a livebacterial product.
 176. The method of claim 173 or 174, wherein thebacterial product is a lyophilized or freeze-dried bacterial product.177. The method of any of claims 173-176, wherein the bacterial productis a bacterial product isolated from a human subject.
 178. The method ofany of claims 173-176, wherein the bacterial product is a bacterialproduct isolated from a non-human subject.
 179. The method of any ofclaims 158-178, wherein the composition is administered orally.
 180. Themethod of any of claims 158-179, wherein the composition furthercomprises a prebiotic.
 181. The method of claim 180, wherein theprebiotic comprises one or more of galactooligosacchararide, lactulose,lactitol, erythritol, isomalt, polyglycitol, and succinate.
 182. Themethod of claim 180, wherein the prebiotic comprises one or more ofgalactooligosacchararide, lactulose, succinate, and lactitol.
 183. Themethod of claim 180, wherein the prebiotic is a resistant starch. 184.The method of claim 180, wherein the prebiotic is a potato starch. 185.The method of any of claims 180-184, wherein the prebiotic comprises oneor more of a digestible and a non-digestible oligosaccharide.
 186. Themethod of any of claims 180-185, wherein at least 10 grams of prebioticis administered to the subject.
 187. The method of any of claims180-186, wherein the ratio of the colony forming units of thePhascolarctobacterium faecium or Ruminococcus bromii to grams ofprebiotic in the composition is 1000:1-10000:1.
 188. The method of anyof claims 158-187, wherein the composition is administered together witha food of the food allergy.
 189. The method of claim 188, wherein thecomposition is administered prior to the food.
 190. The method of claim189, wherein the composition is administered at most 24 hours prior tothe food.
 191. The method of claim 190, wherein the composition isadministered at most 12 hours prior to the food.
 192. The method ofclaim 191, wherein the composition is administered at most 6 hours priorto the food.
 193. The method of claim 188, wherein the composition isadministered after the food.
 194. The method of claim 193, wherein thecomposition is administered at most 24 hours after the food.
 195. Themethod of claim 194, wherein the composition is administered at most 12hours after the food.
 196. The method of claim 195, wherein thecomposition is administered at most 6 hours after the food.
 197. Themethod of any of claims 147-196, further comprising providing animmunotherapy to the subject.
 198. The method of claim 197, wherein theimmunotherapy is an oral immunotherapy.
 199. The method of claim 198,wherein the immunotherapy is an epicutaneous immunotherapy.
 200. Themethod of any of claims 197-199, wherein the immunotherapy is providedprior to administering the composition to the subject.
 201. The methodof any of claims 197-199, wherein the immunotherapy is provided afteradministering the composition to the subject.
 202. A freeze-dried orlyophilized composition comprising Phascolarctobacterium faecium andRuminococcus bromii.
 203. The composition of claim 202, wherein thecomposition further comprises a pharmaceutical excipient.
 204. Thecomposition of claim 202 or 203, wherein the composition is formulatedfor oral administration.
 205. The composition of any of claims 202-204,wherein the composition comprises between 1×10³ and 1×10¹⁵ CFU ofPhascolarctobacterium faecium.
 206. The composition of any of claims202-205, the composition comprises between 1×10³ and 1×10¹⁵ CFU ofRuminococcus bromii.
 207. The composition of any of claims 202-206,wherein the Phascolarctobacterium faecium and Ruminococcus bromii makeup at least 75% of all bacteria in the composition.
 208. The compositionof claim 207, wherein the Phascolarctobacterium faecium and Ruminococcusbromii make up at least 95% of all bacteria in the composition.
 209. Thecomposition of claim 208, wherein the Phascolarctobacterium faecium andRuminococcus bromii make up about 100% of all bacteria in thecomposition.
 210. The composition of any of claims 202-209, wherein thecomposition comprises (i) between 1×10³ and 1×10¹⁵ CFU ofPhascolarctobacterium faecium and (ii) between 1×10³ and 1×10¹⁵ CFU ofRuminococcus bromii.
 211. The composition of any of claims 202-210,wherein the composition does not comprise more than a contaminatingamount of any other bacteria.
 212. The composition of any of claims202-211, wherein the composition does not comprise a detectable amountof any other bacteria.
 213. The composition of any of claims 202-212,further comprising one or more microparticles.
 214. The composition ofclaim 213, wherein the Phascolarctobacterium faecium and Ruminococcusbromii are encapsulated within the one or more microparticles.
 215. Thecomposition of claim 214, wherein the Phascolarctobacterium faecium andRuminococcus bromii are not encapsulated within the one or moremicroparticles.
 216. The composition of any of claims 202-215, furthercomprising a prebiotic.
 217. The composition of claim 216, wherein theprebiotic comprises one or more of galactooligosacchararide, lactulose,lactitol, erythritol, isomalt, polyglycitol, and succinate.
 218. Thecomposition of claim 216, wherein the prebiotic comprises one or more ofgalactooligosacchararide, lactulose, succinate, and lactitol.
 219. Themethod of claim 216, wherein the prebiotic is a resistant starch. 220.The method of claim 216, wherein the prebiotic is a potato starch. 221.The composition of any of claims 202-220, wherein the prebioticcomprises one or more of a digestible and a non-digestibleoligosaccharide.
 222. A tablet, capsule, or powder comprising thecomposition of any of claims 202-221.
 223. A method for determining anOTU abundance score in a subject, the method comprising: (a) obtaining afecal sample from the subject; (b) sequencing nucleic acid from thefecal sample; and (c) identifying OTUs in the fecal sample; wherein theOTUs comprise at least 20 of the OTUs of Table
 1. 224. The method ofclaim 223, wherein the OTUs comprise at least 30 of the OTUs of Table 1.225. The method of claim 224, wherein the OTUs comprise at least 40 ofthe OTUs of Table
 1. 226. The method of claim 225, wherein the OTUscomprise at least 50 of the OTUs of Table
 1. 227. The method of claim226, wherein the OTUs comprise at least 60 of the OTUs of Table
 1. 228.The method of claim 225, wherein the OTUs comprise all of the OTUs ofTable
 1. 229. The method of any of claims 223-228, further comprisingcomparing the OTUs in the fecal sample with OTUs from a control orhealthy sample.
 230. The method of claim 229, further comprisingidentifying the subject as having a food allergy if the OTU abundancescore is increased relative to an OTU abundance score of a control orhealthy sample.
 231. The method of claim 229 or 230, wherein the foodallergy is an allergy to one or more of peanuts, tree nuts, shellfish,soy, egg, fish, mustard, oats, olives, corn, rice, pineapple, wheat,gluten, milk, sesame, garbanzo beans, bananas, kiwi, avocado, mangos,melons, carrots, cucumber, apples, squash, and crab.
 232. A method fortreating a subject determined to have a decreased OTU abundance scorerelative to a control or healthy sample, the method comprising providingto the subject a composition comprising a therapeutically effectiveamount of (a) Phascolarctobacterium faecium or (b) Ruminococcus bromii,wherein the OTU abundance score was determined using at least 20 of theOTUs of Table
 1. 233. The method of claim 232, wherein the OTU abundancescore was determined using at least 30 of the OTUs of Table
 1. 234. Themethod of claim 233, wherein the OTU abundance score was determinedusing at least 40 of the OTUs of Table
 1. 235. The method of claim 234,wherein the OTU abundance score was determined using at least 50 of theOTUs of Table
 1. 236. The method of claim 235, wherein the OTU abundancescore was determined using at least 60 of the OTUs of Table
 1. 237. Themethod of claim 236, wherein the OTU abundance score was determinedusing all of the OTUs of Table
 1. 238. The method of any of claims232-237, wherein the composition comprises Phascolarctobacteriumfaecium.
 239. The method of claim 238, wherein the composition comprisesbetween 1×10³ and 1×10¹⁵ CFU of Phascolarctobacterium faecium.
 240. Themethod of any of claims 232-239, wherein the composition comprisesRuminococcus bromii.
 241. The method of claim 240, wherein thecomposition comprises between 1×10³ and 1×10¹⁵ CFU of Ruminococcusbromii.
 242. The method of any of claims 232-241, wherein thePhascolarctobacterium faecium or Ruminococcus bromii make up at least75% of all bacteria in the composition.
 243. The method of claim 242,wherein the Phascolarctobacterium faecium or Ruminococcus bromii make upat least 95% of all bacteria in the composition.
 244. The method ofclaim 243, wherein the Phascolarctobacterium faecium or Ruminococcusbromii make up about 100% of all bacteria in the composition.
 245. Themethod of any of claims 232-244, wherein the composition comprisesPhascolarctobacterium faecium and Ruminococcus bromii.
 246. The methodof claim 245, wherein the composition comprises (i) between 1×10³ and1×10¹⁵ CFU of Phascolarctobacterium faecium and (ii) between 1×10³ and1×10¹⁵ CFU of Ruminococcus bromii.
 247. The method of any of claims232-246, wherein the composition does not comprise more than acontaminating amount of any other bacteria.
 248. The method of any ofclaims 232-247, wherein the composition does not comprise a detectableamount of any other bacteria.
 249. The method of any of claims 232-248,wherein the composition further comprises one or more microparticles.250. The method of claim 249, wherein the Phascolarctobacterium faeciumor Ruminococcus bromii are encapsulated within the one or moremicroparticles.
 251. The method of claim 250, wherein thePhascolarctobacterium faecium or Ruminococcus bromii are notencapsulated within the one or more microparticles.
 252. The method ofany of claims 232-251, wherein the composition comprises a bacterialproduct comprising the Phascolarctobacterium faecium or Ruminococcusbromii.
 253. The method of claim 252, wherein the bacterial productcomprises Phascolarctobacterium faecium and Ruminococcus bromii. 254.The method of claim 252 or 253, wherein the bacterial product is a livebacterial product.
 255. The method of claim 252 or 253, wherein thebacterial product is a lyophilized or freeze-dried bacterial product.256. The method of any of claims 252-255, wherein the bacterial productis a bacterial product isolated from a human subject.
 257. The method ofany of claims 252-255, wherein the bacterial product is a bacterialproduct isolated from a non-human subject.
 258. The method of any ofclaims 252-257, wherein the composition is administered orally.
 259. Themethod of any of claims 232-258, wherein the composition furthercomprises a prebiotic.
 260. The method of claim 259, wherein theprebiotic comprises one or more of galactooligosacchararide, lactulose,lactitol, erythritol, isomalt, polyglycitol, and succinate.
 261. Themethod of claim 259, wherein the prebiotic comprises one or more ofgalactooligosacchararide, lactulose, succinate, and lactitol.
 262. Themethod of claim 259, wherein the prebiotic is a resistant starch. 263.The method of claim 259, wherein the prebiotic is a potato starch. 264.The method of any of claims 258-263, wherein the prebiotic comprises oneor more of a digestible and a non-digestible oligosaccharide.
 265. Themethod of any of claims 258-264, wherein at least 10 grams of prebioticis administered to the subject.
 266. The method of any of claims258-265, wherein the ratio of the colony forming units of thePhascolarctobacterium faecium or Ruminococcus bromii to grams ofprebiotic in the composition is 1000:1-10000:1.